Anti-adrenomedullin (adm) antibody or anti-adm antibody fragment or anti-adm non-ig scaffold for prevention or reduction of organ dysfunction or organ failure in a patient having a chronic or acute disease or acute condition

ABSTRACT

Subject matter of the present invention is an anti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold for use in therapy of a chronical or acute disease or acute condition of a patient for prevention or reduction of organ dysfunction or organ failure. In a preferred embodiment subject matter of the invention is an anti-ADM antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold for use in therapy of a chronical or acute disease or acute condition of a patient for prevention or reduction of kidney dysfunction or kidney failure or liver dysfunction or liver failure.

FIELD OF THE INVENTION

Subject matter of the present invention is an anti-adrenomedullin (ADM)antibody or an anti-adrenomedullin antibody fragment or an anti-ADMnon-Ig scaffold for use in therapy of a chronical or acute disease oracute condition of a patient for prevention or reduction of organdysfunction or organ failure.

In a preferred embodiment subject matter of the invention is an anti-ADMantibody or an anti-adrenomedullin antibody fragment or an anti-ADMnon-Ig scaffold for use in therapy of a chronical or acute disease oracute condition of a patient for prevention or reduction of kidneydysfunction or kidney failure or for prevention or reduction of liverdysfunction or liver failure.

BACKGROUND

The peptide adrenomedullin (ADM) was described for the first time in1993 (Kitamura, K., et al., “Adrenomedullin: A Novel Hypotensive PeptideIsolated From Human Pheochromocytoma”, Biochemical and BiophysicalResearch Communications, Vol. 192 (2), pp. 553-560 (1993)) as a novelhypotensive peptide comprising 52 amino acids, which had been isolatedfrom a human pheochromocytome; SEQ ID No.: 21. In the same year, cDNAcoding for a precursor peptide comprising 185 amino acids and thecomplete amino acid sequence of this precursor peptide were alsodescribed. The precursor peptide, which comprises, inter alia, a signalsequence of 21 amino acids at the N-terminus, is referred to as“preproadrenomedullin” (pre-proADM). In the present description, allamino acid positions specified usually relate to the pre-proADM whichcomprises the 185 amino acids. The peptide adrenomedullin (ADM) is apeptide which comprises 52 amino acids (SEQ ID NO: 21) and whichcomprises the amino acids 95 to 146 of pre-proADM, from which it isformed by proteolytic cleavage. To date, substantially only a fewfragments of the peptide fragments formed in the cleavage of thepre-proADM have been more exactly characterized, in particular thephysiologically active peptides adrenomedullin (ADM) and “PAMP”, apeptide comprising 20 amino acids (22-41) which follows the 21 aminoacids of the signal peptide in pre-proADM. The discovery andcharacterization of ADM in 1993 triggered intensive research activity,the results of which have been summarized in various review articles, inthe context of the present description, reference being made inparticular to the articles to be found in an issue of “Peptides” devotedto ADM in particular (Editorial, Takahashi, K., “Adrenomedullin: from apheochromocytoma to the eyes”, Peptides, Vol. 22, p. 1691 (2001)) and(Eto, T., “A review of the biological properties and clinicalimplications of adrenomedullin and proadrenomedullin N-terminal 20peptide (PAMP), hypotensive and vasodilating peptides”, Peptides, Vol.22, pp. 1693-1711 (2001)). A further review is (Hinson, et al.,“Adrenomedullin, a Multifunctional Regulatory Peptide”, EndocrineReviews, Vol. 21(2), pp. 138-167 (2000)). In the scientificinvestigations to date, it has been found, inter alia, that ADM may beregarded as a polyfunctional regulatory peptide. It is released into thecirculation in an inactive form extended by glycine (Kitamura, K., etal., “The intermediate form of glycine-extended adrenomedullin is themajor circulating molecular form in human plasma”, Biochem. Biophys.Res. Commun., Vol. 244(2), pp. 551-555 (1998). Abstract Only). There isalso a binding protein (Pio, R., et al., “Complement Factor H is aSerum-binding Protein for adrenomedullin, and the Resulting ComplexModulates the Bioactivities of Both Partners”, The Journal of BiologicalChemistry, Vol. 276(15), pp. 12292-12300 (2001)) which is specific forADM and probably likewise modulates the effect of ADM. Thosephysiological effects of ADM as well as of PAMP which are of primaryimportance in the investigations to date were the effects influencingblood pressure.

Hence, ADM is an effective vasodilator, and thus it is possible toassociate the hypotensive effect with the particular peptide segments inthe C-terminal part of ADM. It has furthermore been found that theabovementioned further physiologically active peptide PAMP formed frompre-proADM likewise exhibits a hypotensive effect, even if it appears tohave an action mechanism differing from that of ADM (cf. in addition tothe abovementioned review articles (Eto, T., “A review of the biologicalproperties and clinical implications of adrenomedullin andproadrenomedullin N-terminal 20 peptide (PAMP), hypotensive andvasodilating peptides”, Peptides, Vol. 22, pp. 1693-1711 (2001)) and(Hinson, et al., “Adrenomedullin, a Multifunctional Regulatory Peptide”,Endocrine Reviews, Vol. 21(2), pp. 138-167 (2000)) also (Kuwasako, K.,et al., “Purification and characterization of PAMP-12 (PAMP-20) inporcine adrenal medulla as a major endogenous biologically activepeptide”, FEBS Lett, Vol. 414(1), pp. 105-110 (1997). Abstract only),(Kuwasaki, K., et al., “Increased plasma proadrenomedullin N-terminal 20peptide in patients with essential hypertension”, Ann. Clin. Biochem.,Vol. 36 (Pt. 5), pp. 622-628 (1999). Abstract only) or (Tsuruda, T., etal., “Secretion of proadrenomedullin N-terminal 20 peptide from culturedneonatal rat cardiac cells”, Life Sci., Vol. 69(2), pp. 239-245 (2001).Abstract only) and EP-A2 0 622 458). It has furthermore been found thatthe concentrations of ADM which can be measured in the circulation andother biological liquids are, in a number of pathological states,significantly above the concentrations to be found in healthy controlpersons. Thus, the ADM level in patients with congestive heart failure,myocardial infarction, kidney diseases, hypertensive disorders, Diabetesmellitus, in the acute phase of shock and in sepsis and septic shock aresignificantly increased, although to different extents. The PAMPconcentrations are also increased in some of said pathological states,but the plasma levels are lower relative to ADM ((Eto, T., “A review ofthe biological properties and clinical implications of adrenomedullinand proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive andvasodilating peptides”, Peptides, Vol. 22, pp. 1693-1711 (2001)); page1702). It is furthermore known that unusually high concentrations of ADMare to be observed in sepsis, and the highest concentrations in septicshock (cf. (Eto, T., “A review of the biological properties and clinicalimplications of adrenomedullin and proadrenomedullin N-terminal 20peptide (PAMP), hypotensive and vasodilating peptides”, Peptides, Vol.22, pp. 1693-1711 (2001)) and (Hirata, et al., “Increased CirculatingAdrenomedullin, a Novel Vasodilatory Peptide, in Sepsis”, Journal ofClinical Endocrinology and Metabolism, Vol. 81(4), pp. 1449-1453(1996)), (Ehlenz, K., et al., “High levels of circulating adrenomedullinin severe illness: Correlation with C-reactive protein and evidenceagainst the adrenal medulla as site of origin”, Exp Clin EndocrinolDiabetes, Vol. 105, pp. 156-162 (1997)), (Tomoda, Y., et al.,“Regulation of adrenomedullin secretion from cultured cells”, Peptides,Vol. 22, pp. 1783-1794 (2001)), (Ueda, S., et al., “Increased PlasmaLevels of Adrenomedullin in Patients with Systemic Inflammatory ResponseSyndrome”, Am. J. Respir. Crit. Care Med., Vol. 160, pp. 132-136 (1999))and (Wang, P., “Adrenomedullin and cardiovascular responses in sepsis”,Peptides, Vol. 22, pp. 1835-1840 (2001))).

Known in the art is further a method for identifying adrenomedullinimmunoreactivity in biological liquids for diagnostic purposes and, inparticular within the scope of sepsis diagnosis, cardiac diagnosis andcancer diagnosis. According to the invention, the midregional partialpeptide of the proadrenomedullin, which contains amino acids (45-92) ofthe entire preproadrenomedullin, is measured, in particular, with animmunoassay which works with at least one labeled antibody thatspecifically recognizes a sequence of the mid-proADM. (WO2004/090546).

WO-A1 2004/097423 describes the use of an antibody againstadrenomedullin for diagnosis, prognosis, and treatment of cardiovasculardisorders. Treatment of diseases by blocking the ADM receptor are alsodescribed in the art, (e.g. WO-A1 2006/027147, PCT/EP2005/012844) saiddiseases may be sepsis, septic shock, cardiovascular diseases,infections, dermatological diseases, endocrinological diseases,metabolic diseases, gastroenterological diseases, cancer, inflammation,hematological diseases, respiratory diseases, muscle skeleton diseases,neurological diseases, urological diseases.

It is reported for the early phase of sepsis that ADM improves heartfunction and the blood supply in liver, spleen, kidney and smallintestine. ADM-neutralizing antibodies neutralize the before mentionedeffects during the early phase of sepsis (Wang, P., “Adrenomedullin andcardiovascular responses in sepsis”, Peptides, Vol. 22, pp. 1835-1840(2001).

In the later phase of sepsis, the hypodynamical phase of sepsis, ADMconstitutes a risk factor that is strongly associated with the mortalityof patients in septic shock. (Schütz et al., “Circulating Precursorlevels of endothelin-1 and adrenomedullin, two endothelium-derived,counteracting substances, in sepsis”, Endothelium, 14:345-351, (2007)).Methods for the diagnosis and treatment of critically ill patients, e.g.in the very late phasis of sepsis, and the use of endothelin andendothelin agonists with vasoconstrictor activity for the preparation ofmedicaments for the treatment of critically ill patients have beendescribed in WO-A1 2007/062676. It is further described in WO-A12007/062676 to use, in place of endothelin and/or endothelin agonists,or in combination therewith, adrenomedullin antagonists, i.e. moleculeswhich prevent or attenuate the vasodilating action of adrenomedullin,e.g. by blocking its relevant receptors, or substances preventing thebinding of adrenomedullin to its receptor (e.g. specific binders as e.g.antibodies binding to adrenomedullin and blocking its receptor bindingssites; “immunological neutralization”). Such use, or combined use,including a subsequent or preceding separate use, has been described incertain cases to be desirable for example to improve the therapeuticsuccess, or to avoid undesirable physiological stress or side effects.Thus, it is reported that neutralizing ADM antibodies may be used forthe treatment of sepsis in the late stage of sepsis.

Administration of ADM in combination with ADM-binding-Protein-1 isdescribed for treatment of sepsis and septic shock in the art. It isassumed that treatment of septic animals with ADM andADM-binding-Protein-1 prevents transition to the late phase of sepsis.It has to be noted that in a living organism ADM binding protein(complement factor H) is present in the circulation of said organism inhigh concentrations (Pio et al.: Identification, characterization, andphysiological actions of complement factor H as an Adrenomedullinbinding Protein present in Human Plasma; Microscopy Res. and Technique,55:23-27 (2002) and Martinez et al.; Mapping of theAdrenomedullin-Binding domains in Human Complement factor H; HypertensRes Vol. 26, Suppl (2003), S56-59).

In accordance with the invention the ADM-binding-Protein-1 may be alsoreferred to as ADM-binding-Protein-1 (complement factor H).

Prevention or reduction of organ dysfunction or organ failure is veryimportant when treating a patient having a chronic or acute disease oracute condition, especially a life threatening disease or condition.

DESCRIPTION OF THE INVENTION

Subject matter of the present invention is an anti-adrenomedullin (ADM)antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Igscaffold for use in therapy of a chronic or acute disease or acutecondition of a patient for prevention or reduction of organ dysfunctionor organ failure. In a preferred embodiment subject matter of theinvention is an anti-ADM antibody or an anti-adrenomedullin antibodyfragment or anti-ADM non-Ig scaffold for use in therapy of a chronic oracute disease or acute condition of a patient for prevention orreduction of kidney dysfunction or kidney failure or liver dysfunctionor liver failure. Said organ may be selected from the group comprisingheart, kidney, liver, lungs, pancreas, small intestines and spleen.Anti-ADM antibody or an anti-adrenomedullin antibody fragment oranti-ADM non-Ig scaffold may be administered at any point of time beforethe occurrence of dysfunction or failure or after the occurrence ofdysfunction or failure.

“Organ dysfunction” denotes a condition or a state of health where anorgan does not perform its expected function. “Organ failure” denotes anorgan dysfunction to such a degree that normal homeostasis cannot bemaintained without external clinical intervention.

By contrast, organ function represents the expected function of therespective organ within physiologic ranges. The person skilled in theart is aware of the respective function of an organ during medicalexamination. Therefore, in the following only basic information inregard to particular organs within the scope of the invention isprovided:

The heart is a chambered muscular organ that pumps blood received fromthe veins into the arteries. Thereby the heart is maintaining the flowof blood through the circulatory system entirely to supply oxygen to thebody.

The kidneys is a pair of organs that function to maintain adequate waterand electrolyte balance, they regulate acid-base concentration, andfurther filter the blood of metabolic wastes, which are afterwardsexcreted as urine.

The liver is a large organ that secretes bile and is active in theformation of certain blood proteins and in the metabolism ofcarbohydrates, fats, and proteins.

The lungs are functioning to remove carbon dioxide from the blood andprovide it with oxygen.

The pancreas secretes pancreatic juice into the duodenum and insulin,glucagon, and somatostatin into the bloodstream.

The small intestines are the part of the digestive tract, in which theprocess of digestion is practically completed. It is narrow andcontorted, and consists of three parts, the duodenum, jejunum, andileum.

The spleen plays important roles in regard to erythrocytes—the red bloodcells—and the immune system. Specifically, the spleen removes old redblood cells and holds a reserve of blood in case of hemorrhagic shockwhile also recycling iron. Further, it metabolizes hemoglobin removedfrom senescent erythrocytes. The globin portion of hemoglobin isdegraded to its constitutive amino acids, and the heme portion ismetabolized to bilirubin, which is subsequently shuttled to the liverfor its removal. In addition, the spleen synthesizes antibodies in itswhite pulp and removes antibody-coated bacteria along withantibody-coated blood cells by way of blood and lymph node circulation.

It should be emphasized that the provided anti-adrenomedullin (ADM)antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Igscaffold are intended by the present invention to be applied for sake ofprevention or reduction of organ dysfunction and organ failure, and thusare not necessarily intended for any methods of primary treatment orfirst line treatment to the chronic or acute disease or acute conditionitself, which therefore can be termed as underlying disease(s). Thismeans the present invention does not provide for a therapy ofhealing/curing e.g. infections, cancer, or tumors located in therespective organ, but for resuscitating the respective organ towardsphysiologic function. Accordingly, the therapy for a chronic or acutedisease or acute condition of a patient within the scope of theinvention is related to any kind of organ insufficiency, or poor organfunction as an acute event.

Specifically according to the invention it has to be understood that incase of any organ dysfunction or organ failure of pancreas that is e.g.due to diabetes mellitus, the herein provided anti-ADM antibody,anti-ADM antibody fragment, or anti-ADM non-Ig scaffold are not intendedfor first line treatment of diabetes, but for resuscitating pancreas'physiologic function.

Specifically according to the invention it has to be understood that incase of any organ dysfunction or organ failure of e.g. pancreas, lung,liver, kidney, spleen, small intestine, heart that is due to canceroustumors or cancer, the herein provided anti-ADM antibody, anti-ADMantibody fragment, or anti-ADM non-Ig scaffold are not intended forfirst line treatment of cancerous tumors or cancer in the respectiveorgan, but for resuscitating the respective organ's physiologicfunction.

The patient group(s) addressed by the instant invention can be definedas set out below.

In the following, clinical criteria are mentioned for respective organsthat are prone to dysfunction or failure, and thus represent the patientgroup(s) of having a chronic or acute disease or acute condition inaccordance with the invention:

The criteria orientate on the clinical SOFA score.

The SOFA system was created in a consensus meeting of the EuropeanSociety of Intensive Care Medicine in 1994 and further revised in 1996.

The SOFA is a six-organ dysfunction/failure score measuring multipleorgan failure daily. Each organ is graded from 0 (normal) to 4 (the mostabnormal), providing a daily score of 0 to 24 points. The objective ofthe SOFA is to create a simple, reliable, and continuous score forclinical staff.

Sequential assessment of organ dysfunction during the first few days ofintensive care unit (ICU) or hospital admission is a good indicator ofprognosis. Both the mean and highest SOFA scores are particularly usefulpredictors of outcome.

SOFA score 0 1 2 3 4 Respiration PaO₂/FIO₂ (mmHg) >400 <400 <300 <200<100 SaO₂/FIO₂ 221-301 142-220  67-141 <67 Coagulation Platelets >150<150 <100 <50 <20 10³/mm³ Liver Bilirubin <1.2 1.2-1.9 2.0-5.9 6.0-11.9 >12.0 (mg/dL) Cardiovascular^(b) Hypotension No MAP DopamineDopamine >5 Dopamine >15 hypotension <70 </=5 or or or dobutaminenorepinephrine norepinephrine (any) </=0.1 >0.1 CNS Glasgow Coma 1513-14 10-12 6-9 <6 Score Renal Creatinine <1.2 1.2-1.9 2.0-3.4 3.5-4.9or >5.0 or <200 (mg/dL) <500 or urine output (mL/d) MAP, mean arterialpressure; CNS, central nervous system; SaO₂, peripheral arterial oxygensaturation. ^(a)PaO₂/FIO₂ ratio was used preferentially. If notavailable, the SaO₂/FIO₂ ratio was used; ^(b)vasoactive mediationsadministered for at least 1 hr (dopamine and norepinephrine μg/kg/min).

REFERENCES FOR SOFA SCORE

-   1. Jones A E, Trzeciak S, Kline J A. The Sequential Organ Failure    Assessment score for predicting outcome in patients with severe    sepsis and evidence of hypoperfusion at the time of emergency    department presentation. Crit Care Med. 2009 May; 37(5):1649-54.-   2. Ferreira F L, Bota D P, Bross A, Mélot C, Vincent J L. Serial    evaluation of the SOFA score to predict outcome in critically ill    patients. JAMA. 2001 Oct. 10; 286(14):1754-8.-   3. Vincent J L, Moreno R, Takala J, Willatts S, De Mendonça A,    Bruining H, Reinhart C K, Suter P M, Thijs L G. The SOFA    (Sepsis-related Organ Failure Assessment) score to describe organ    dysfunction/failure. On behalf of the Working Group on    Sepsis-Related Problems of the European Society of Intensive Care    Medicine. Intensive Care Med. 1996 July; 22(7):707-10.

In a specific embodiment the patient group pursuant to the invention ishaving as lower threshold at least one SOFA score, being it 1 for onethe clinical criteria respiration, or liver, or coagulation, orcardiovascular, or CNS, or renal on day of admission to hospital orIntensive Care Unit (ICU). Thus, said patient group is in need oftherapeutic intervention pursuant to the invention, and thus in need forprevention or reduction of organ dysfunction or organ failure

In another specific embodiment the patient group pursuant to theinvention is having as lower threshold at least two SOFA scores, beingit 1 each for the clinical criteria respiration, and/or liver, and/orcoagulation, and/or cardiovascular, and/or CNS, and/or renal on day ofadmission to hospital or Intensive Care Unit (ICU). Thus, said patientgroup is in need of therapeutic intervention pursuant to the invention,and thus in need for prevention or reduction of organ dysfunction ororgan failure.

In another specific embodiment the patient group pursuant to theinvention is having as lower threshold at least three SOFA scores, beingit 1 each for the clinical criteria respiration, and/or liver, and/orcoagulation, and/or cardiovascular, and/or CNS, and/or renal on day ofadmission to hospital or Intensive Care Unit (ICU). Thus, said patientgroup is in need of therapeutic intervention pursuant to the invention,and thus in need for prevention or reduction of organ dysfunction ororgan failure.

In another specific embodiment the patient group pursuant to theinvention is having as lower threshold at least four SOFA scores, beingit 1 each for the clinical criteria respiration, and/or liver, and/orcoagulation, and/or cardiovascular, and/or CNS, and/or renal on day ofadmission to hospital or Intensive Care Unit (ICU). Thus, said patientgroup is in need of therapeutic intervention pursuant to the invention,and thus in need for prevention or reduction of organ dysfunction ororgan failure.

In another specific embodiment the patient group in need for preventionor reduction of renal organ dysfunction or renal organ failure pursuantto the invention is having a renal SOFA score of at least 1, or of 2, orof 3, or of 4.

In another specific embodiment the patient group in need for preventionor reduction of liver organ dysfunction or liver organ failure pursuantto the invention is having a liver SOFA score of at least 1, or of 2, orof 3, or of 4.

In another specific embodiment the patient group in need for preventionor reduction of heart organ dysfunction or heart organ failure pursuantto the invention is having a cardiovascular SOFA score of at least 1, orof 2, or of 3, or of 4.

In another specific embodiment the patient group in need for preventionor reduction of lung organ dysfunction or lung organ failure pursuant tothe invention is having a respiratory SOFA score of at least 1, or of 2,or of 3, or of 4.

Independent of the initial score, generally an increase in SOFA scoreduring the first 48 hours in the ICU or in the hospital predicts amortality rate of at least 50%.

Thus, in another specific embodiment the patient group in need oftherapeutic intervention for organ dysfunction/failure in accordancewith invention is characterized by having at least one SOFA scoreincreased within the initial 48 hours after admission to hospital orICU.

Patient Group—Kidney Dysfunction/Failure

In the following, said clinical criteria denote the patient group(s) forkidney dysfunction/failure:

-   -   Patients at risk for kidney dysfunction/failure: GFR        decrease >25%, serum creatinine increased 1.5 times or urine        production of <0.5 ml/kg/hr for 6 hours    -   Patients with present kidney injury: GFR decrease >50%, doubling        of creatinine or urine production <0.5 ml/kg/hr for 12 hours    -   Patients with kidney failure: GFR decrease >75%, tripling of        creatinine or creatinine >355 μmol/l (with a rise of >44) (>4        mg/dl) or urine output below 0.3 ml/kg/hr for 24 hours    -   Patients with loss of kidney function: persistent acute kidney        injury (AKI) or complete loss of kidney function for more than 4        weeks    -   end-stage renal disease: complete loss of kidney function for        more than 3 months.

Patient Group—Liver Dysfunction/Failure

The patient group for liver dysfunction/failure is characterized by alower threshold of Bilirubin of >1.2 mg/dL, preferably >1.9 mg/dL, morepreferably >5.9 mg/dL.

Oxygen Depletion

The person skilled in the art is aware that sepsis is associated withmitochondrial dysfunction, which inevitably leads to impaired oxygenconsumption and ultimately to sepsis-induced multiple organ failure.

This holds especially true for raised tissue oxygen tensions in septicpatients, suggesting reduced ability of the organs to use oxygen.Because ATP production by mitochondrial oxidative phosphorylationaccounts for more than 90% of total oxygen consumption mitochondrialdysfunction may directly results in organ failure, possibly due tonitric oxide, which is known to inhibit mitochondrial respiration invitro and is produced in excess in sepsis.

Therefore, in a very specific embodiment of the invention the anti-ADMantibody or the anti-ADM antibody fragment or anti-ADM non-Ig scaffoldare particularly intended to be used in methods of prevention for organdysfunction and failure in SIRS, sepsis, severe sepsis, shock and septicshock patients.

Oxygene depletion may be also caused by ischemic events as e.g. by passsurgery.

The anti-ADM antibody or the anti-ADM antibody fragment or anti-ADMnon-Ig scaffold may be also administered preventively before the patientexhibits any signs of dysfunction or failure of an organ. This might bethe case if the patient has a chronic or acute disease or acutecondition where dysfunction or failure problems may be expected, e.g.comprising severe infections as e.g. meningitis, Systemic inflammatoryResponse-Syndrome (SIRS,) sepsis; other diseases as diabetes, cancer,acute and chronic vascular diseases as e.g. heart failure, myocardialinfarction, stroke, atherosclerosis; shock as e.g. septic shock andorgan dysfunction as e.g. kidney dysfunction, liver dysfunction,burnings, surgery, traumata, poisoning. The anti-ADM antibody or theanti-ADM antibody fragment or anti-ADM non-Ig scaffold may be alsoadministered preventively or therapeutically before, or during or afterchemotherapy. The same applies for surgeries where ischemic damages mayoccur to certain organs which may result in dysfunction or failure of anorgan. Preventively means before an organ damage occurs andtherapeutically means that an organ damage has been already occurred.Especially useful is the antibody or fragment or scaffold according tothe present invention for reducing the risk of organ dysfunction orfailure during sepsis and septic shock, i.e. late phases of sepsis.

Acute disease or acute conditions may be selected from the group but arenot limited to the group comprising severe infections as e.g.meningitis, Systemic inflammatory Response-Syndrom (SIRS), or sepsis;other diseases as diabetes, cancer, acute and chronic vascular diseasesas e.g. heart failure, myocardial infarction, stroke, atherosclerosis;shock as e.g. septic shock and organ dysfunction as e.g. kidneydysfunction, liver dysfunction, burnings, surgery, traumata, poisoning,damages induced by chemotherapy. Especially useful is the antibody orfragment or scaffold according to the present invention for reducing therisk of mortality during sepsis and septic shock, i.e. late phases ofsepsis.

In the following clinical criteria for SIRS, sepsis, severe sepsis,septic shock will be defined.

1) Systemic Inflammatory Host Response (SIRS) Characterized by at LeastTwo of the Following Symptoms

-   -   patients exhibit hypotension (mean arterial pressure is <65 mm        Hg)    -   elevated serum lactate level being >4 mmol/L    -   blood glucose >7.7 mmol/L (in absence of diabetes)    -   central venous pressure is not within the range 8-12 mm Hg    -   urine output is <0.5 mL×kg⁻¹×hr⁻¹    -   central venous (superior vena cava) oxygen saturation is <70% or        mixed venous <65%    -   heart rate is >90 beats/min    -   temperature <36° C. or >38° C.    -   respiratory rate >20/min    -   white cell count <4 or >12×10⁹/L (leucocytes); >10% immature        neutrophils

2) Sepsis

Following at least two of the symptoms mentioned under 1), andadditionally a clinical suspicion of new infection, being:

-   -   cough/sputum/chest pain    -   abdominal pain/distension/diarrhoea    -   line infection    -   endocarditis    -   dysuria    -   headache with neck stiffness    -   cellulitis/wound/joint infection    -   positive microbiology for any infection

3) Severe Sepsis

Provided that sepsis is manifested in patient, and additionally aclinical suspicion of any organ dysfunction, being:

-   -   blood pressure systolic <90/mean; <65 mmHG    -   lactate >2 mmol/L    -   Bilirubine >34 μmol/L    -   urine output <0.5 mL/kg/h for 2 h    -   creatinine >177 μmol/L    -   platelets <100×10⁹/L    -   SpO₂>90% unless O₂ given

4) Septic Shock

At least one sign of end-organ dysfunction as mentioned under 3) ismanifested. Septic shock is indicated, if there is refractoryhypotension that does not respond to treatment and intravenous systemicfluid administration alone is insufficient to maintain a patient's bloodpressure from becoming hypotensive also provides for an administrationof an anti-ADM antibody or an anti-ADM antibody fragment or an anti-ADMnon-Ig scaffold in accordance with the present invention.

Thus, acute disease or acute conditions may be selected from the groupbut are not limited to the group comprising severe infections as e.g.meningitis, Systemic inflammatory Response-Syndrome (SIRS), or sepsis;other diseases as diabetes, cancer, acute and chronic vascular diseasesas e.g. heart failure, myocardial infarction, stroke, atherosclerosis;shock as e.g. septic shock and organ dysfunction as e.g. kidneydysfunction, liver dysfunction, burnings, surgery, traumata, poisoning,damages induced by chemotherapy. Especially useful is the antibody orfragment or scaffold according to the present invention for reducing therisk of mortality during sepsis and septic shock, i.e. late phases ofsepsis.

In one embodiment of the present invention the patient is not sufferingfrom SIRS, a severe infection, sepsis, shock as e.g. septic shock. Saidsevere infection denotes e.g. meningitis, Systemic inflammatoryResponse-Syndrome (SIRS), sepsis, severe sepsis, and shock as e.g.septic shock. In this regard, a severe sepsis is characterized in thatsepsis is manifested in said patient, and additionally a clinicalsuspicion of any organ dysfunction is present, being it:

-   -   blood pressure systolic <90/mean; <65 mmHG    -   lactate >2 mmol/L    -   Bilirubine >34 μmol/L    -   urine output <0.5 mL/kg/h for 2 h    -   creatinine >177 μmol/L    -   platelets <100×10⁹/L    -   SpO₂>90% unless O₂ given

In another specific embodiment said acute disease or acute condition isnot sepsis, severe sepsis or is not SIRS or is not shock, or septicshock.

In another embodiment said acute disease or acute condition is notsepsis.

In another embodiment said acute disease or acute condition is selectedfrom the group comprising meningitis, diabetes, cancer, acute andchronic vascular diseases as e.g. heart failure, myocardial infarction,stroke, atherosclerosis; shock as e.g. septic shock and organdysfunction as e.g. kidney dysfunction, liver dysfunction, burnings,surgery, traumata, poisoning, damages induced by chemotherapy.

It should be emphasized that the herein provided anti-ADM antibody,anti-ADM antibody fragment or anti-ADM non-Ig scaffold is not intendedfor first line treatment of any organ associated diseases such asnephroliths, renal cancer, nephritis, liver cirrhosis, fatty liver,hepatic cancer, or e.g. hepatitis. The anti-ADM antibody or an anti-ADMantibody fragment or an anti-ADM non-Ig scaffold in accordance with theinvention are intended to prevent from or address a malfunction in therespective organ's physiologic function.

The organ protecting effect of the anti-ADM antibody or the anti-ADMantibody fragment or anti-ADM non-Ig scaffold is thus supporting theprimary therapy of said chronic or acute disease or acute condition. Incase of a chronic or acute disease or acute condition like a severeinfection, SIRS, sepsis or the like the primary therapy would be e.g.the administration of antibiotics. The anti-ADM antibody or the anti-ADMantibody fragment or anti-ADM non-Ig scaffold would protect the organand would help to prevent worsening of the critical condition of saidpatient until the e.g. antibiotic administration takes effect. As beforementioned the anti-ADM antibody or the anti-ADM antibody fragment oranti-ADM non-Ig scaffold may be administered in a preventive way or in atherapeutic way, this means in order to prevent dysfunction or failureproblems or in order to reduce organ dysfunction when dysfunctionproblems are present in a said patient.

In another specific embodiment of the invention the expression “theanti-ADM antibody or the anti-ADM antibody fragment or anti-ADM non-Igscaffold may be administered in a preventive way or in a therapeuticway” denotes systemic administration to a patient.

In one embodiment of the invention an Anti-adrenomedullin (ADM) antibodyor an anti-ADM antibody fragment or anti-ADM non-Ig scaffold is to beused in combination with vasopressors e.g. catecholamine wherein saidcombination is for use in therapy of a chronic or acute disease or acutecondition of a patient for protecting an organ of said patient.

Subject matter of the invention in one specific embodiment is, thus, ananti-ADM (ADM) antibody or an anti-ADM antibody fragment or anti-ADMnon-Ig scaffold for use in therapy of a patient in need of anadministration of vasopressors e.g. of catecholamine administration.

Furthermore, in one embodiment of the invention an anti-adrenomedullin(ADM) antibody or an anti-adrenomedullin antibody fragment or ananti-ADM non-Ig scaffold is to be used in combination with fluidsadministered intravenously, wherein said combination is for use intherapy of a patient having a chronic or acute disease or acutecondition of a patient for protecting the organs of said patient. Itshould be emphasized that said fluids to be administered intravenouslyare administered systemically.

In one embodiment of the invention said patient having a chronic oracute disease or condition being in need for protecting its organs ischaracterized by the need of said patient to get intravenous fluids.

It should be noted that in accordance with the invention administrationof fluids is in the meaning of systemic administration of fluids.

Subject matter of the invention in one specific embodiment is, thus, ananti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibodyfragment or anti-ADM non-Ig scaffold for use in therapy of a patient inneed of intravenous fluids. Even with restoration of adequate bloodpressure and normal or supranormal cardiac output, signs of tissuehypoperfusion may persist. This is often called “distributive shock” andmay be related to maldistribution and blood flow at the regional ormicrovascular level and/or a cellular inability to utilize oxygendespite adequate oxygen delivery. It is clinically important that tissuehypoperfusion be recognized, despite what may appear to be “normal”blood pressures, and should trigger timely intervention. According tothe present invention such an intervention is the administration of ananti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibodyfragment or anti-ADM non-Ig scaffold for use in therapy of a chronic oracute disease or acute condition of a patient for prevention orreduction of organ dysfunction.

An antibody according to the present invention is a protein includingone or more polypeptides substantially encoded by immunoglobulin genesthat specifically binds an antigen. The recognized immunoglobulin genesinclude the kappa, lambda, alpha (IgA), gamma (IgG₁, IgG₂, IgG₃, IgG₄),delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as wellas the myriad immunoglobulin variable region genes. Full-lengthimmunoglobulin light chains are generally about 25 Kd or 214 amino acidsin length. Full-length immunoglobulin heavy chains are generally about50 Kd or 446 amino acid in length. Light chains are encoded by avariable region gene at the NH2-terminus (about 110 amino acids inlength) and a kappa or lambda constant region gene at the COOH-terminus.Heavy chains are similarly encoded by a variable region gene (about 116amino acids in length) and one of the other constant region genes.

The basic structural unit of an antibody is generally a tetramer thatconsists of two identical pairs of immunoglobulin chains, each pairhaving one light and one heavy chain. In each pair, the light and heavychain variable regions bind to an antigen, and the constant regionsmediate effector functions. Immunoglobulins also exist in a variety ofother forms including, for example, Fv, Fab, and (Fab′)₂, as well asbifunctional hybrid antibodies and single chains (e.g., Lanzavecchia etal., Eur. J. Immunol. 17:105, 1987; Huston et al., Proc. Natl. Acad.Sci. U.S.A., 85:5879-5883, 1988; Bird et al., Science 242:423-426, 1988;Hood et al., Immunology, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller andHood, Nature 323:15-16, 1986). An immunoglobulin light or heavy chainvariable region includes a framework region interrupted by threehypervariable regions, also called complementarity determining regions(CDR's) (see, Sequences of Proteins of Immunological Interest, E. Kabatet al., U.S. Department of Health and Human Services, 1983). As notedabove, the CDRs are primarily responsible for binding to an epitope ofan antigen. An immune complex is an antibody, such as a monoclonalantibody, chimeric antibody, humanized antibody or human antibody, orfunctional antibody fragment, specifically bound to the antigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody can be joined to human constant segments, such askappa and gamma 1 or gamma 3. In one example, a therapeutic chimericantibody is thus a hybrid protein composed of the variable orantigen-binding domain from a mouse antibody and the constant oreffector domain from a human antibody, although other mammalian speciescan be used, or the variable region can be produced by moleculartechniques. Methods of making chimeric antibodies are well known in theart, e.g., see U.S. Pat. No. 5,807,715. A “humanized” immunoglobulin isan immunoglobulin including a human framework region and one or moreCDRs from a non-human (such as a mouse, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor” and the human immunoglobulin providing the framework istermed an “acceptor.” In one embodiment, all the CDRs are from the donorimmunoglobulin in a humanized immunoglobulin. Constant regions need notbe present, but if they are, they must be substantially identical tohuman immunoglobulin constant regions, i.e., at least about 85-90%, suchas about 95% or more identical. Hence, all parts of a humanizedimmunoglobulin, except possibly the CDRs, are substantially identical tocorresponding parts of natural human immunoglobulin sequences. A“humanized antibody” is an antibody comprising a humanized light chainand a humanized heavy chain immunoglobulin. A humanized antibody bindsto the same antigen as the donor antibody that provides the CDRs. Theacceptor framework of a humanized immunoglobulin or antibody may have alimited number of substitutions by amino acids taken from the donorframework. Humanized or other monoclonal antibodies can have additionalconservative amino acid substitutions which have substantially no effecton antigen binding or other immunoglobulin functions. Exemplaryconservative substitutions are those such as gly, ala; val, ile, leu;asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Humanizedimmunoglobulins can be constructed by means of genetic engineering(e.g., see U.S. Pat. No. 5,585,089). A human antibody is an antibodywherein the light and heavy chain genes are of human origin. Humanantibodies can be generated using methods known in the art. Humanantibodies can be produced by immortalizing a human B cell secreting theantibody of interest. Immortalization can be accomplished, for example,by EBV infection or by fusing a human B cell with a myeloma or hybridomacell to produce a trioma cell. Human antibodies can also be produced byphage display methods (see, e.g., Dower et al., PCT Publication No.WO91/17271; McCafferty et al., PCT Publication No. WO92/001047; andWinter, PCT Publication No. WO92/20791), or selected from a humancombinatorial monoclonal antibody library (see the Morphosys website).Human antibodies can also be prepared by using transgenic animalscarrying a human immunoglobulin gene (for example, see Lonberg et al.,PCT Publication No. WO93/12227; and Kucherlapati, PCT Publication No.WO91/10741).

Thus, the anti-ADM antibody or an anti-adrenomedullin antibody fragmentmay have the formats known in the art. Examples are human antibodies,monoclonal antibodies, humanized antibodies, chimeric antibodies,CDR-grafted antibodies. In a preferred embodiment antibodies accordingto the present invention are recombinantly produced antibodies as e.g.IgG, a typical full-length immunoglobulin, or antibody fragmentscontaining at least the F-variable domain of heavy and/or light chain ase.g. chemically coupled antibodies (fragment antigen binding) includingbut not limited to Fab-fragments including Fab minibodies, single chainFab antibody, monovalent Fab antibody with epitope tags, e.g. Fab-V5Sx2;bivalent Fab (mini-antibody) dimerized with the CH3 domain; bivalent Fabor multivalent Fab, e.g. formed via multimerization with the aid of aheterologous domain, e.g. via dimerization of dHLX domains, e.g.Fab-dHLX-FSx2; F(ab′)₂-fragments, scFv-fragments, multimerizedmultivalent or/and multispecific scFv-fragments, bivalent and/orbispecific diabodies, BITE® (bispecific T-cell engager), trifunctionalantibodies, polyvalent antibodies, e.g. from a different class than G;single-domain antibodies, e.g. nanobodies derived from camelid or fishimmunoglobulines and numerous others.

Furthermore, in one embodiment of the invention an anti-Adrenomedullin(ADM) antibody or an anti-adrenomedullin antibody fragment or ananti-ADM non-Ig scaffold is monospecific.

Monospecific anti-Adrenomedullin (ADM) antibody or monospecificanti-adrenomedullin antibody fragment or monospecific anti-ADM non-Igscaffold means that said antibody or antibody fragment or non-Igscaffold binds to one specific region encompassing at least 5 aminoacids within the target ADM.

Monospecific anti-Adrenomedullin (ADM) antibody or monospecificanti-adrenomedullin antibody fragment or monospecific anti-ADM non-Igscaffold are anti-Adrenomedullin (ADM) antibodies or anti-adrenomedullinantibody fragments or anti-ADM non-Ig scaffolds that all have affinityfor the same antigen.

In another preferred embodiment of the invention an anti-Adrenomedullin(ADM) antibody or an anti-adrenomedullin antibody fragment or ananti-ADM non-Ig scaffold is monospecific.

Monospecific anti-Adrenomedullin (ADM) antibody or monospecificanti-adrenomedullin antibody fragment or monospecific anti-ADM non-Igscaffold means that said antibody or antibody fragment or non-Igscaffold binds to one specific region encompassing at least 4 aminoacids within the target ADM.

In another special embodiment the anti-ADM antibody or the antibodyfragment binding to ADM is a monospecific antibody. Monospecific meansthat said antibody or antibody fragment binds to one specific regionencompassing at least 5 amino acids within the target ADM.

In another special and preferred embodiment the anti-ADM antibody or theantibody fragment binding to ADM is a monospecific antibody.Monospecific means that said antibody or antibody fragment binds to onespecific region encompassing at least 4 amino acids within the targetADM.

Monospecific antibodies or fragments are antibodies or fragments thatall have affinity for the same antigen. Monoclonal antibodies aremonospecific, but monospecific antibodies may also be produced by othermeans than producing them from a common germ cell.

In addition to anti-ADM antibodies other biopolymer scaffolds are wellknown in the art to complex a target molecule and have been used for thegeneration of highly target specific biopolymers. Examples are aptamers,spiegelmers, anticalins and conotoxins. For illustration of antibodyformats please see FIGS. 1 a, 1 b and 1 c.

In a preferred embodiment the anti-ADM antibody format is selected fromthe group comprising Fv fragment, scFv fragment, Fab fragment, scFabfragment, F(ab)₂ fragment and scFv-Fc Fusion protein. In anotherpreferred embodiment the antibody format is selected from the groupcomprising scFab fragment, Fab fragment, scFv fragment andbioavailability optimized conjugates thereof, such as PEGylatedfragments. One of the most preferred formats is the scFab format.

Non-Ig scaffolds may be protein scaffolds and may be used as antibodymimics as they are capable to bind to ligands or antigenes. Non-Igscaffolds may be selected from the group comprising tetranectin-basednon-Ig scaffolds (e.g. described in US 2010/0028995), fibronectinscaffolds (e.g. described in EP 1266 025; lipocalin-based scaffolds((e.g. described in WO 2011/154420); ubiquitin scaffolds (e.g. describedin WO 2011/073214), transferring scaffolds (e.g. described in US2004/0023334), protein A scaffolds (e.g. described in EP 2231860),ankyrin repeat based scaffolds (e.g. described in WO 2010/060748),microproteins preferably microproteins forming a cystine knot) scaffolds(e.g. described in EP 2314308), Fyn SH3 domain based scaffolds (e.g.described in WO 2011/023685) EGFR-A-domain based scaffolds (e.g.described in WO 2005/040229) and Kunitz domain based scaffolds (e.g.described in EP 1941867).

In one embodiment of the invention anti-ADM antibodies according to thepresent invention may be produced as follows:

A Balb/c mouse was immunized with 100 μg ADM-Peptide(antigen)-BSA-Conjugate at day 0 and 14 (emulsified in 100 μl completeFreund's adjuvant) and 50 μg at day 21 and 28 (in 100 μl incompleteFreund's adjuvant). Three days before the fusion experiment wasperformed, the animal received 50 μg of the conjugate dissolved in 100μl saline, given as one intraperitoneal and one intra-venous injection.

Spenocytes from the immunized mouse and cells of the myeloma cell lineSP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C.After washing, the cells were seeded in 96-well cell culture plates.Hybrid clones were selected by growing in HAT medium [RPMI 1640 culturemedium supplemented with 20% fetal calf serum and HAT-Supplement]. Aftertwo weeks the HAT medium is replaced with HT Medium for three passagesfollowed by returning to the normal cell culture medium.

The cell culture supernatants were primary screened for antigen specificIgG antibodies three weeks after fusion. The positive testedmicrocultures were transferred into 24-well plates for propagation.After retesting, the selected cultures were cloned and recloned usingthe limiting-dilution technique and the isotypes were determined. (seealso Lane, R. D. (1985). A short-duration polyethylene glycol fusiontechnique for increasing production of monoclonal antibody-secretinghybridomas. J. Immunol. Meth. 81: 223-228; Ziegler, B. et al. (1996)Glutamate decarboxylase (GAD) is not detectable on the surface of ratislet cells examined by cytofluorometry and complement-dependentantibody-mediated cytotoxicity of monoclonal GAD antibodies, Horm.Metab. Res. 28: 11-15).

Antibodies may be produced by means of phage display according to thefollowing procedure:

The human naive antibody gene libraries HALT/8 were used for theisolation of recombinant single chain F-Variable domains (scFv) againstadrenomedullin peptide. The antibody gene libraries were screened with apanning strategy comprising the use of peptides containing a biotin taglinked via two different spacers to the adrenomedullin peptide sequence.A mix of panning rounds using non-specifically bound antigen andstreptavidin bound antigen were used to minimize background ofnon-specific binders. The eluted phages from the third round of panninghave been used for the generation of monoclonal scFv expressing E. colistrains. Supernatant from the cultivation of these clonal strains hasbeen directly used for an antigen ELISA testing (see also Hust, M.,Meyer, T., Voedisch, B., Rülker, T., Thie, H., El-Ghezal, A., Kirsch, M.I., Schütte, M., Helmsing, S., Meier, D., Schirrmann, T., Dübel, S.,2011. A human scFv antibody generation pipeline for proteome research.Journal of Biotechnology 152, 159-170; Schütte, M., Thullier, P., Pelat,T., Wezler, X., Rosenstock, P., Hinz, D., Kirsch, M. I., Hasenberg, M.,Frank, R., Schirrmann, T., Gunzer, M., Hust, M., Dübel, S., 2009.Identification of a putative Crf splice variant and generation ofrecombinant antibodies for the specific detection of Aspergillusfumigatus. PLoS One 4, e6625)

Humanization of murine antibodies may be conducted according to thefollowing procedure:

For humanization of an antibody of murine origin the antibody sequenceis analyzed for the structural interaction of framework regions (FR)with the complementary determining regions (CDR) and the antigen. Basedon structural modeling an appropriate FR of human origin is selected andthe murine CDR sequences are transplanted into the human FR. Variationsin the amino acid sequence of the CDRs or FRs may be introduced toregain structural interactions, which were abolished by the speciesswitch for the FR sequences. This recovery of structural interactionsmay be achieved by random approach using phage display libraries or viadirected approach guided by molecular modeling. (Almagro J C, FranssonJ., 2008. Humanization of antibodies. Front Biosci. 2008 Jan. 1;13:1619-33.)

In a preferred embodiment the ADM antibody format is selected from thegroup comprising Fv fragment, scFv fragment, Fab fragment, scFabfragment, F(ab)₂ fragment and scFv-Fc Fusion protein. In anotherpreferred embodiment the antibody format is selected from the groupcomprising scFab fragment, Fab fragment, scFv fragment andbioavailability optimized conjugates thereof, such as PEGylatedfragments. One of the most preferred formats is scFab format.

In another preferred embodiment, the anti-ADM antibody, anti-ADMantibody fragment, or anti-ADM non-Ig scaffold is a full lengthantibody, antibody fragment, or non-Ig scaffold.

In a preferred embodiment the anti-ADM antibody or ananti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold isdirected to and can bind to an epitope of at least 5 amino acids inlength contained in ADM.

In a more preferred embodiment the anti-ADM antibody or ananti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold isdirected to and can bind to an epitope of at least 4 amino acids inlength contained in ADM.

In a preferred embodiment of the present invention said anti-ADMantibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Igscaffold binds to a region of ADM that is located in the N-terminal part(aa 1-21) of adrenomedullin, (see FIG. 2).

In another preferred embodiment said antibody or an anti-adrenomedullinantibody fragment or anti-ADM non-Ig scaffold recognizes and binds tothe N-terminal end (aa1) of adrenomedullin. N-terminal end means thatthe amino acid 1, that is “Y” of SEQ ID No. 21 or 23; is mandatory forbinding. The antibody or fragment or scaffold would neither bindN-terminal extended nor N-terminal modified adrenomedullin norN-terminal degraded adrenomedullin.

In one specific embodiment of the invention the anti-Adrenomedullin(ADM) antibody or anti-ADM antibody fragment binding to adrenomedullinor anti-ADM non-Ig scaffold binding to adrenomedullin is provided foruse in therapy of an acute disease or acute condition of a patientwherein said antibody or antibody fragment or non-Ig scaffold binds to aregion of preferably at least 4, or at least 5 amino acids within thesequence of aa 1-42 of mature human ADM:

SEQ ID No: 24 YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA.

In one specific embodiment of the invention the anti-Adrenomedullin(ADM) antibody or anti-ADM antibody fragment binding to adrenomedullinor anti-ADM non-Ig scaffold binding to adrenomedullin is provided foruse in therapy of an acute disease or acute condition of a patientwherein said antibody or fragment or scaffold binds to a region ofpreferably at least 4, or at least 5 amino acids within the sequence ofaa 1-21 of mature human ADM:

SEQ ID No: 23 YRQSMNNFQGLRSFGCRFGTC.

In another specific embodiment pursuant to the invention the hereinprovided anti-ADM antibody or anti-ADM antibody fragment or anti-ADMnon-Ig scaffold does not bind to the C-terminal portion of ADM, i.e. theaa 43-52 of ADM (SEQ ID NO: 25)

(SEQ ID NO: 25) PRSKISPQGY-NH2.

In one specific embodiment it is preferred to use an anti-ADM antibodyor an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffoldaccording to the present invention, wherein said anti-adrenomedullinantibody or said anti-adrenomedullin antibody fragment or non-Igscaffold is an ADM stabilizing antibody or an adrenomedullin stabilizingantibody fragment or an adrenomedullin stabilizing non-Ig scaffold thatenhances the half life (t_(1/2); half retention time) of adrenomedullinin serum, blood, plasma at least 10%, preferably at least 50%, morepreferably >50%, most preferably >100%.

The half life (half retention time) of ADM may be determined in humanplasma in absence and presence of an ADM stabilizing antibody or anadrenomedullin stabilizing antibody fragment or an adrenomedullinstabilizing non-Ig scaffold, respectively, using an immunoassay for thequantification of ADM.

The following steps may be conducted:

-   -   ADM may be diluted in human citrate plasma in absence and        presence of an ADM stabilizing antibody or an adrenomedullin        stabilizing antibody fragment or an adrenomedullin stabilizing        non-Ig scaffold, respectively, and may be incubated at 24° C.    -   Aliquots are taken at selected time points (e.g. within 24        hours) and degradation of ADM may be stopped in said aliquots by        freezing at −20° C.    -   The quantity of ADM may be determined by an hADM immunoassay        directly, if the selected assay is not influenced by the        stabilizing antibody. Alternatively, the aliquot may be treated        with denaturing agents (like HCl) and, after clearing the sample        (e.g. by centrifugation) the pH can be neutralized and the        ADM-quantified by an ADM immunoassay. Alternatively,        non-immunoassay technologies (e.g. rpHPLC) can be used for        ADM-quantification    -   The half life of ADM is calculated for ADM incubated in absence        and presence of an ADM stabilizing antibody or an adrenomedullin        stabilizing antibody fragment or an adrenomedullin stabilizing        non-Ig scaffold, respectively,    -   The enhancement of half life is calculated for the stabilized        ADM in comparison to ADM that has been incubated in absence of        an ADM stabilizing antibody or an adrenomedullin stabilizing        antibody fragment or an adrenomedullin stabilizing non-Ig        scaffold.

A two-fold increase of the half life of ADM is an enhancement of halflife of 100%.

Half Life (half retention time) is defined as the period over which theconcentration of a specified chemical or drug takes to fall to halfbaseline concentration in the specified fluid or blood.

An assay that may be used for the determination of the Half life (halfretention time) of adrenomedullin in serum, blood, plasma is describedin Example 3.

For some diseases blocking of ADM may be beneficial to a certain extent.However, it might also be detrimental if ADM is totally neutralized as acertain amount of ADM may be required for several physiologicalfunctions. In many reports it was emphasized that the administration ofADM may be beneficial in certain diseases. In contrast thereto in otherreports ADM was reported as being life threatening when administered incertain conditions.

In a preferred embodiment said anti-ADM antibody, anti-ADM antibodyfragment or anti-ADM non-Ig scaffold is a non-neutralizing antibody,fragment or scaffold. A neutralizing anti-ADM antibody, anti-ADMantibody fragment or anti-ADM non-Ig scaffold would block thebioactivity of ADM to nearly 100%, to at least more than 90%, preferablyto at least more than 95%.

In contrast, a non-neutralizing anti-ADM antibody, or anti-ADM antibodyfragment or anti-ADM non-Ig scaffold blocks the bioactivity of ADM lessthan 100%, preferably to less than 95%, preferably to less than 90%,more preferred to less than 80% and even more preferred to less than50%. This means that the residual bioactivity of ADM bound to thenon-neutralizing anti-ADM antibody, or anti-ADM antibody fragment oranti-ADM non-Ig scaffold would be more than 0%, preferably more than 5%,preferably more than 10%, more preferred more than 20%, more preferredmore than 50%.

In this context (a) molecule(s), being it an antibody, or an antibodyfragment or a non-Ig scaffold with “non-neutralizing anti-ADM activity”,collectively termed here for simplicity as “non-neutralizing” anti-ADMantibody, antibody fragment, or non-Ig scaffold, that e.g. blocks thebioactivity of ADM to less than 80%, is defined as

-   -   a molecule or molecules binding to ADM, which upon addition to a        culture of an eukaryotic cell line, which expresses functional        human recombinant ADM receptor composed of CRLR (calcitonin        receptor like receptor) and RAMP3 (receptor-activity modifying        protein 3), reduces the amount of cAMP produced by the cell line        through the action of parallel added human synthetic ADM        peptide, wherein said added human synthetic ADM is added in an        amount that in the absence of the non-neutralizing antibody to        be analyzed, leads to half-maximal stimulation of cAMP        synthesis, wherein the reduction of cAMP by said molecule(s)        binding to ADM takes place to an extent, which is not more than        80%, even when the non-neutralizing molecule(s) binding to ADM        to be analyzed is added in an amount, which is 10-fold more than        the amount, which is needed to obtain the maximal reduction of        cAMP synthesis obtainable with the non-neutralizing antibody to        be analyzed.

The same definition applies to the other ranges; 95%, 90%, 50% etc.

In a specific embodiment according to the present invention an anti-ADMantibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Igscaffold is used, wherein said antibody or an anti-adrenomedullinantibody fragment blocks the bioactivity of ADM to less than 80%,preferably less than 50% (of baseline values). This means the antibody,antibody fragment or non-Ig scaffold pursuant to the invention blocksthe bioactivity of ADM to not more than 80%, or not more than 50%,respectively. By implication, this means at least 20% residual ADMbioactivity or at least 50% residual ADM bioactivity is present whenusing an antibody, antibody fragment or non-Ig scaffold in accordancewith the invention. It has been understood that said limited blocking ofthe bioactivity of ADM occurs even at excess concentration of theantibody, fragment or scaffold, meaning an excess of the antibody,fragment or scaffold in relation to ADM. Said limited blocking is anintrinsic property of the ADM binder itself. This means that saidantibody, fragment or scaffold has a maximal inhibition of 80% or 50%respectively. In a preferred embodiment said anti-ADM antibody, anti-ADMantibody fragment or anti-ADM non-Ig scaffold would block thebioactivity of ADM to at least 5%. By implication, this meansapproximately 95% residual ADM bioactivity is present.

The bioactivity is defined as the effect that a substance takes on aliving organism or tissue or organ or functional unit in vivo or invitro (e.g. in an assay) after its interaction. In case of ADMbioactivity this may be the effect of ADM in a human recombinantAdrenomedullin receptor cAMP functional assay. Thus, according to thepresent invention bioactivity is defined via an Adrenomedullin receptorcAMP functional assay. The following steps may be performed in order todetermine the bioactivity of ADM in such an assay:

-   -   Dose response curves are performed with ADM in said human        recombinant Adrenomedullin receptor cAMP functional assay.    -   The ADM-concentration of half-maximal cAMP stimulation may be        calculated.    -   At constant half-maximal cAMP-stimulating ADM-concentrations        dose response curves (up to 100 μg/ml final concentration) are        performed by an ADM stabilizing antibody or an adrenomedullin        stabilizing antibody fragment or an adrenomedullin stabilizing        non-Ig scaffold, respectively,

A maximal inhibition in said ADM bioassay of 50% means that saidanti-ADM antibody or said anti-ADM antibody fragment or said anti-ADMnon-Ig scaffold, respectively, blocks the bioactivity to 50% of baselinevalues. A maximal inhibition in said ADM bioassay of 80% means that saidanti-ADM antibody or said anti-adrenomedullin antibody fragment or saidanti-adrenomedullin non-Ig scaffold, respectively, blocks thebioactivity of ADM to 80%. This is in the sense of blocking the ADMbioactivity to not more than 80%. This means approximately 20% residualADM bioactivity remains present.

However, by the present specification and in the above context theexpression “blocks the bioactivity of ADM” in relation to the hereindisclosed anti-ADM antibodies, anti-ADM antibody fragments, and anti-ADMnon-Ig scaffolds should be understood as mere decreasing the bioactivityof ADM from 100% to 20% remaining ADM bioactivity at maximum, preferablydecreasing the ADM bioactivity from 100% to 50% remaining ADMbioactivity; but in any case there is ADM bioactivity remaining that canbe determined as detailed above.

The bioactivity of ADM may be determined in a human recombinantAdrenomedullin receptor cAMP functional assay (Adrenomedullin Bioassay)according to Example 2.

In a preferred embodiment a modulating anti-ADM antibody or a modulatinganti-ADM antibody fragment is used in the treatment of sepsis. Amodulating anti-ADM antibody or a modulating anti-adrenomedullinantibody fragment enhances the bioactivity of ADM in the early phase ofsepsis and reduces the damaging effects of ADM in the late phase ofsepsis. A “modulating” anti-ADM antibody or a modulatinganti-adrenomedullin antibody fragment is an antibody that enhances thehalf life (t_(1/2) half retention time) of adrenomedullin in serum,blood, plasma at least 10%, preferably at least, 50%, morepreferably >50%, most preferably >100% and blocks the bioactivity of ADMto less than 80%, preferably less than 50%.

In a preferred embodiment a modulating anti-ADM antibody or a modulatinganti-adrenomedullin antibody fragment or a modulatinganti-adrenomedullin non-Ig scaffold is used in therapy of a chronic oracute disease or acute condition of a patient for stabilizing thecirculation, in particular for stabilizing the systemic circulation.

Such a modulating antibody or a modulating anti-adrenomedullin antibodyfragment or a modulating anti-adrenomedullin non-Ig scaffold may beespecially useful in the treatment of sepsis. A modulating antibody or amodulating anti-adrenomedullin antibody fragment or a modulatinganti-adrenomedullin non-Ig scaffold enhances the bioactivity of ADM inthe early phase of sepsis and reduces the damaging effects of ADM in thelate phase of sepsis.

In a preferred embodiment the anti-adrenomedullin antibody or ananti-adrenomedullin antibody fragment or anti-adrenomedullin non-Igscaffold is directed to and can bind to an epitope of at least 5 aminoacids in length contained in ADM, preferably in human ADM.

In a more preferred embodiment the anti-adrenomedullin antibody or ananti-adrenomedullin antibody fragment or anti-adrenomedullin non-Igscaffold is directed to and can bind to an epitope of at least 4 aminoacids in length contained in ADM, preferably in human ADM.

A “modulating” anti-ADM antibody or a modulating anti-adrenomedullinantibody fragment or a modulating anti-adrenomedullin non-Ig scaffold isan antibody or an anti-adrenomedullin antibody fragment or non-Igscaffold that enhances the half life (t_(1/2) half retention time) ofadrenomedullin in serum, blood, plasma at least 10%, preferably atleast, 50%, more preferably >50%, most preferably >100% and blocks thebioactivity of ADM to less than 80%, preferably less than 50% andwherein said anti-ADM antibody, anti-ADM antibody fragment or anti-ADMnon-Ig scaffold would block the bioactivity of ADM to at least 5%. Thesevalues related to half-life and blocking of bioactivity have to beunderstood in relation to the before-mentioned assays in order todetermine these values. This is in the sense of blocking the ADM of notmore than 80% or not more than 50%, respectively.

Such a modulating anti-adrenomedullin antibody or a modulatinganti-adrenomedullin antibody fragment or a modulatinganti-adrenomedullin non-Ig scaffold offers the advantage that the dosingof the administration is facilitated. The combination of partiallyblocking or partially reducing Adrenomedullin bioactivity and increaseof the in vivo half life (increasing the Adrenomedullin bioactivity)leads to beneficial simplicity of anti-adrenomedullin antibody or ananti-adrenomedullin antibody fragment or anti-adrenomedullin non-Igscaffold dosing. In a situation of excess of endogenous Adrenomedullin(maximal stimulation, late sepsis phase, shock, hypodynamic phase) theactivity lowering effect is the major impact of the antibody or fragmentor scaffold, limiting the (negative) effect of Adrenomedullin. In caseof low or normal endogenous Adrenomedullin concentrations, thebiological effect of anti-adrenomedullin antibody or ananti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is acombination of lowering (by partially blocking) and increase byincreasing the Adrenomedullin half life. If the half life effect isstronger than the net blocking effect, the biological activity ofendogenous Adrenomedullin is beneficially increased in early phases ofSepsis (low Adrenomedullin, hyperdynamic phase). Thus, thenon-neutralizing and modulating anti-antibody or anti-adrenomedullinantibody fragment or anti-adrenomedullin non-Ig scaffold acts like anADM bioactivity buffer in order to keep the bioactivity of ADM within acertain physiological range.

Thus, the dosing of the antibody/fragment/scaffold in e.g. sepsis may beselected from an excessive concentration, because both Sepsis phases(early and late) benefit from excessive anti-ADM antibody or an anti-ADMantibody fragment or anti-ADM non-Ig scaffold treatment in case of amodulating effect. Excessive means: The anti-adrenomedullin antibody oran anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffoldconcentration is higher than endogenous Adrenomedullin during late phase(shock) of e.g. sepsis. This means, in case of a modulating antibody ormodulating fragment or modulating scaffold dosing in sepsis may be asfollows:

The concentration of Adrenomedullin in septic shock is 226+/−66 fmol/ml(Nishio et al., “Increased plasma concentrations of adrenomedullincorrelate with relaxation of vascular tone in patients with septicshock.”, Crit Care Med. 1997, 25(6):953-7), an equimolar concentrationof antibody or fragment or scaffold is 42.5 μg/1 blood, (based on 6 lblood volume/80 kg body weight) 3.2 μg/kg body weight. Excess means atleast double (mean) septic shock Adrenomedullin concentration, atleast >3 μg anti-adrenomedullin antibody or an anti-adrenomedullinantibody fragment or anti-ADM non-Ig Scaffold/kg body weight, preferredat least 6.4 μg anti-adrenomedullin antibody or an anti-adrenomedullinantibody fragment anti-ADM non-Ig Scaffold/kg body weight. Preferred >10μg/kg, more preferred >20 μg/kg, most preferred >100 μganti-adrenomedullin antibody or an anti-adrenomedullin antibody fragmentor anti-ADM non-Ig scaffold/kg body weight. This may apply to othersevere and acute conditions than septic shock as well.

In a specific embodiment of the invention the antibody is a monoclonalantibody or a fragment thereof. In one embodiment of the invention theanti-ADM antibody or the anti-ADM antibody fragment is a human orhumanized antibody or derived therefrom. In one specific embodiment oneor more (murine) CDR's are grafted into a human antibody or antibodyfragment.

Subject matter of the present invention in one aspect is a humanCDR-grafted antibody or antibody fragment thereof that binds to ADM,wherein the human CDR-grafted antibody or antibody fragment thereofcomprises an antibody heavy chain (H chain) comprising

SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST and/or SEQ ID NO: 3TEGYEYDGFDYand/or further comprises an antibody light chain (L chain) comprising:

SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS and/or SEQ ID NO: 6 FQGSHIPYT.

In one specific embodiment of the invention subject matter of thepresent invention is a human monoclonal antibody that binds to ADM or anantibody fragment thereof wherein the heavy chain comprises at least oneCDR selected from the group comprising:

SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDYand wherein the light chain comprises at least one CDR selected from thegroup comprising:

SEQ ID No: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS SEQ ID NO: 6 FQGSHIPYT.

In a more specific embodiment of the invention subject matter of theinvention is a human monoclonal antibody that binds to ADM or anantibody fragment thereof wherein the heavy chain comprises thesequences

SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDYand wherein the light chain comprises the sequences

SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS SEQ ID NO: 6 FQGSHIPYT.

In a very specific embodiment the anti-ADM antibody has a sequenceselected from the group comprising: SEQ ID NO 7, 8, 9, 10, 11, 12, 13and 14.

The anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Igscaffold according to the present invention exhibits an affinity towardshuman ADM in such that affinity constant is greater than 10⁻⁷ M,preferred 10⁻⁸ M, preferred affinity is greater than 10⁻⁹ M, mostpreferred higher than 10⁻¹⁰ M. A person skilled in the art knows that itmay be considered to compensate lower affinity by applying a higher doseof compounds and this measure would not lead out-of-the-scope of theinvention. The affinity constants may be determined according to themethod as described in Example 1.

In a preferred embodiment the anti-ADM antibody or the anti-ADM antibodyfragment or the anti-ADM non-Ig scaffold is used for reducing the riskof mortality during said chronic or acute disease or acute condition ofa patient.

Chronic or acute disease or acute condition according to the presentinvention may be a disease or condition selected from the groupcomprising severe infections as e.g. meningitis, Systemic inflammatoryResponse-Syndrome (SIRS) sepsis; other diseases as diabetes, cancer,acute and chronic vascular diseases as e.g. heart failure, myocardialinfarction, stroke, atherosclerosis; shock as e.g. septic shock andorgan dysfunction as e.g. kidney dysfunction, liver dysfunction,burnings, surgery, traumata, poisoning, damages by chemotherapy.Especially useful is the antibody or fragment or scaffold according tothe present invention for reducing the risk of mortality during sepsisand septic shock, i.e. late phases of sepsis.

In a preferred embodiment the antibody or the antibody fragment is usedfor reducing the risk of mortality during said chronic or acute diseaseof a patient wherein said disease is selected from the group comprisingsepsis, diabetis, cancer, acute anc chronic vascular diseases as e.g.heart failure, shock as e.g. septic shock and organ dysfunction as e.g.kidney dysfunction. Especially useful is the antibody or fragmentaccording to the present invention for reducing the risk of mortalityduring sepsis and septic shock, i.e. late phases of sepsis.

In a preferred embodiment a modulating antibody or a modulatingadrenomedullin antibody fragment is used in the treatment of sepsis. Amodulating antibody enhances the bioactivity of ADM in the early phaseof sepsis and reduces the damaging effects of ADM in the late phase ofsepsis. A “modulating” antibody or an adrenomedullin antibody fragmentis an antibody that enhances the t_(1/2) half retention time ofadrenomedullin in serum, blood, plasma at least 10%, preferably atleast, 50%, more preferably >50%, most preferably >100% and blocks thebioactivity of ADM to less than 80%, preferably less than 50%.

In one embodiment the anti-ADM antibody or an anti-adrenomedullinantibody fragment or anti-ADM non-Ig Scaffold is used in therapy of achronic or acute disease or acute condition of a patient according tothe present invention wherein said patient is an ICU patient. In anotherembodiment the anti-ADM antibody or an anti-adrenomedullin antibodyfragment or anti-ADM non-Ig scaffold is used in therapy of a chronic oracute disease of a patient according to the present invention whereinsaid patient is critically ill. Critically ill means a patient is havinga disease or state in which death is possible or imminent.

Subject of the present invention is further an anti-ADM antibody or ananti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold foruse in therapy of a chronic or acute disease or acute condition of apatient according to the present invention wherein said antibody orfragment or scaffold is to be used in combination of ADM bindingprotein. ADM binding protein is also naturally present in thecirculation of said patient.

It should be emphasized that the term ADM binding protein also denotesADM-binding-protein-1 (complement factor H), which however is not anon-neutralizing and modulating anti-ADM antibody, anti-ADM antibodyfragment, or anti-ADM non-Ig scaffold as in accordance with theinvention.

Subject of the present invention is further an anti-ADM antibody or ananti-ADM antibody fragment or anti-ADM non-Ig scaffold for use intherapy of a chronic or acute disease or acute condition of a patientaccording to the present invention wherein said antibody or fragment orscaffold is to be used in combination with further active ingredients.

Subject matter of the invention is also an anti-adrenomedullin (ADM)antibody or an anti-adrenomedullin antibody fragment or an anti-ADMnon-Ig scaffold to be used in combination with a primary medicamentwherein said combination is for use in therapy of a chronic or acutedisease or acute condition of a patient for prevention or reduction oforgan dysfunction or organ failure in said patient.

Primary medicament means a medicament that acts against the primarycause of said disease or condition said primary medicament may beantibiotics in case of infections.

In a specific embodiment of the before mentioned combinations saidcombinations are to be used in combination with vasopressors e.g.catecholamine wherein said further combination is for use in therapy ofa chronic or acute disease or condition of a patient for prevention orreduction of organ dysfunction or organ failure in said patient.

In one embodiment of the invention said patient having a chronic oracute disease or chronic condition being in need for prevention orreduction of organ dysfunction or organ failure in said patient ischaracterized by the need of the patient to get administration ofvasopressors e.g. catecholamine administration.

Subject matter of the invention in one specific embodiment is, thus, ananti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibodyfragment or an anti-ADM non-Ig scaffold to be used in combination withADM binding protein and/or further active ingredients for use in therapyof a patient in need of a treatment of vasopressors e.g. catecholaminetreatment.

In a specific embodiment of the above mentioned combinations saidcombinations are to be used in combination with fluids administeredintravenously, wherein said combination is for use in therapy of achronic or acute disease or condition of a patient for prevention orreduction of organ dysfunction or organ failure in said patient.

In one embodiment of the invention said patient having a chronic oracute disease or acute condition being in need for prevention orreduction of organ dysfunction or organ failure in said patient ischaracterized by the need of the patient to get intravenous fluids.

Subject matter of the invention in one specific embodiment is, thus, ananti-adrenomedullin (ADM) antibody or an anti-adrenomedullin antibodyfragment or anti-ADM non-Ig scaffold in combination with ADM bindingprotein and/or further active ingredients for use in therapy of apatient in need of intravenous fluids.

In accordance with the invention the ADM-binding-Protein-1 may be alsoreferred to as ADM-binding-Protein-1 (complement factor H).

In one specific embodiment of the invention the anti-adrenomedullin(ADM) antibody or anti-ADM antibody fragment binding to adrenomedullinor anti-ADM non-Ig scaffold binding to adrenomedullin is provided foruse in therapy of an acute disease or acute condition of a patientwherein said antibody or fragment or scaffold is notADM-binding-Protein-1 (complement factor H).

It should be emphasized that the term ADM binding protein also denotesADM-binding-protein-1 (complement factor H), which however is not anon-neutralizing and modulating anti-ADM antibody, anti-ADM antibodyfragment, or anti-ADM non-Ig scaffold as in accordance with theinvention.

Said anti-ADM antibody or an anti-adrenomedullin antibody fragment oranti-ADM non-Ig scaffold or combinations thereof with ADM bindingprotein and/or further active ingredients may be used in combinationwith vasopressors e.g. catecholamine and/or with fluids administeredintravenously for use in a of a chronic or acute disease or acutecondition of a patient for prevention or reduction of organ dysfunctionor organ failure in said patient.

Subject matter of the invention is also an anti-ADM antibody or ananti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffoldaccording to the present invention to be used in combination withTNF-alpha-antibodies. TNF-alpha-antibodies are commercially availablefor the treatment of patients.

Subject of the present invention is further a pharmaceutical formulationcomprising an antibody or fragment or scaffold according to the presentinvention. Subject of the present invention is further a pharmaceuticalformulation according to the present invention wherein saidpharmaceutical formulation is a solution, preferably a ready-to-usesolution.

Said pharmaceutical formulation may be administered intra-muscular. Saidpharmaceutical formulation may be administered intra-vascular. Saidpharmaceutical formulation may be administered via infusion. In anotherembodiment subject of the present invention is further a pharmaceuticalformulation according to the present invention wherein saidpharmaceutical formulation is in a dried state to be reconstitutedbefore use.

In another embodiment subject of the present invention is further apharmaceutical formulation according to the present invention whereinsaid pharmaceutical formulation is in a freeze-dried state.

In another embodiment subject of the present invention is further apharmaceutical formulation according to the present invention whereinsaid pharmaceutical formulation is administered intra-muscular.

In another embodiment subject of the present invention is further apharmaceutical formulation according to the present invention whereinsaid pharmaceutical formulation is administered intra-vascular.

In another embodiment subject of the present invention is further apharmaceutical formulation according to the present invention whereinsaid pharmaceutical formulation is administered via infusion.

It should be emphasized that the pharmaceutical formulation inaccordance with the invention as may be administered intra-muscular,intra-vascular, or via infusion is preferably administered systemicallyto a patient for prevention or reduction of organ dysfunction or organfailure in a patient having a chronic or acute disease or acutecondition.

Therefore, in another embodiment of the present invention thepharmaceutical formulation according to the present invention is to beadministered systemically to a patient for prevention or reduction oforgan dysfunction or organ failure in a patient having a chronic oracute disease or acute condition.

In another specific and preferred embodiment of the present inventionthe pharmaceutical formulation according to the present invention is tobe administered systemically via infusion to a patient for prevention orreduction of organ dysfunction or organ failure in a patient having achronic or acute disease or acute condition.

Further embodiments within the scope of the present invention are setout below:

-   1. Adrenomedullin ADM antibody or an adrenomedullin antibody    fragment for use in therapy of a chronic or acute disease of a    patient for the regulation of liquid balance.-   2. ADM antibody or an adrenomedullin antibody fragment according to    claim 1 wherein the antibody format is selected from the group    comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment,    (Fab)2 fragment and scFv-Fc Fusion protein.-   3. ADM antibody or an adrenomedullin antibody fragment according    claim 1 or 2 wherein said antibody or fragment binds to the    N-terminal part (aa 1-21) of adrenomedullin.-   4. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 3, wherein said antibody or fragment recognizes    and binds to the N-terminal end (aa1) of adrenomedullin.-   5. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 4, wherein said antibody or fragment is an ADM    stabilizing antibody or ADM stabilizing a antibody fragment that    enhances the t_(1/2) half retention time of adrenomedullin in serum,    blood, plasma at least 10%, preferably at least 50%, more    preferably >50%, most preferably >100%.-   6. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 5, wherein said antibody or fragment blocks the    bioactivity of ADM to less than 80%, preferably less than 50%.-   7. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronic or acute disease of a patient according to any    of claims 1 to 6 wherein said disease is selected from the group    comprising sepsis, diabetis, cancer, heart failure, shock and kidney    dysfunction.-   8. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronic or acute disease of a patient according to any    of claims 1 to 7 wherein said patient is an ICU patient.-   9. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronic or acute disease of a patient according to any    of claims 1 to 7 wherein said antibody or fragment is a modulating    antibody or fragment that enhances the t_(1/2) half retention time    of adrenomedullin in serum, blood, plasma at least 10%, preferably    at least 50%, more preferably >50%, most preferably >100% and that    blocks the bioactivity of ADM to less than 80%, preferably less than    50%.-   10. Pharmaceutical formulation comprising an antibody or fragment    according to any of claims 1 to 9.-   11. Pharmaceutical formulation according to claim 10 wherein said    pharmaceutical formulation is a solution, preferably a ready-to-use    solution.-   12. Pharmaceutical formulation according to claim 10 wherein said    pharmaceutical formulation is in a freeze-dried state.-   13. Pharmaceutical formulation according to any of claims 10 to 11,    wherein said pharmaceutical formulation is administered    intra-muscular.-   14. Pharmaceutical formulation according to any of claims 10 to 11,    wherein said pharmaceutical formulation is administered    intra-vascular.-   15. Pharmaceutical formulation according to claim 14, wherein said    pharmaceutical formulation is administered via infusion.

Further embodiments within the scope of the present invention are setout below:

-   1. Adrenomedullin ADM antibody or an adrenomedullin antibody    fragment an ADM non-Ig scaffold for use in therapy of a chronic or    acute disease or acute condition of a patient for the regulation of    fluid balance.-   2. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to claim 1 wherein said ADM antibody or an    adrenomedullin antibody fragment or ADM non-IG scaffold is a    non-neutralizing ADM antibody or a non-neutralizing adrenomedullin    antibody fragment or a non-neutralizing ADM non-IG scaffold.-   3. Adrenomedullin ADM antibody or an adrenomedullin antibody    fragment or an ADM non-Ig scaffold for use in therapy of a chronic    or acute disease or acute condition according to claim 1 or 2 for    preventing or reducing edema in said patient.-   4. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 3 wherein the antibody    format is selected from the group comprising Fv fragment, scFv    fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFv-Fc    Fusion protein.-   5. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 4, wherein said antibody or    fragment or scaffold binds to the N-terminal part (aa 1-21) of    adrenomedullin.-   6. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 5, wherein said antibody or    fragment scaffold recognizes and binds to the N-terminal end (aa1)    of adrenomedullin.-   7. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 6, wherein said antibody or    fragment or scaffold is an ADM stabilizing antibody or ADM    stabilizing antibody fragment or ADM stabilizing non-IG scaffold    that enhances the half life (t_(1/2) half retention time) of    adrenomedullin in serum, blood, plasma at least 10%, preferably at    least 50%, more preferably >50%, most preferably >100%.-   8. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 7, wherein said antibody or    fragment blocks the bioactivity of ADM to less than 80%, preferably    less than 50%.-   9. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold for use in therapy of a chronic or acute disease of a    patient according to any of claims 1 to 8 wherein said disease is    selected from the group comprising SIRS, sepsis, diabetis, cancer,    heart failure, shock and kidney dysfunction-   10. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 9, wherein said antibody or fragment is a human    monoclonal antibody or fragment that binds to ADM or an antibody    fragment thereof wherein the heavy chain comprises the sequences

SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY

-   -   and wherein the light chain comprises the sequences

SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS SEQ ID NO: 6 FQGSHIPYT.

-   11. A human monoclonal antibody or fragment that binds to ADM or an    antibody fragment thereof according to claim 10 wherein said    antibody or fragment comprises a sequence selected from the group    comprising:

SEQ ID NO: 7 (AM-VH-C)QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 8 (AM-VH1)QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 9 (AM-VH2-E40)QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 10 (AM-VH3-T26-E55)QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 11 (AM-VH4-T26-E40-E55)QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 12 (AM-VL-C)DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO: 13 (AM-VL1)DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO: 14 (AM-VL2-E40)DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

-   12. ADM antibody or an adrenomedullin antibody fragment or ADM    non-IG scaffold for use in therapy of a chronic or acute disease of    a patient according to any of claims 1 to 9 wherein said patient is    an ICU patient.-   13. ADM antibody or an adrenomedullin antibody fragment or ADM    non-IG scaffold for use in therapy of a chronic or acute disease of    a patient according to any of claims 1 to 12 wherein said antibody    or fragment or scaffold is a modulating antibody or fragment or    scaffold that enhances the half life (t_(1/2) half retention time)    of adrenomedullin in serum, blood, plasma at least 10%, preferably    at least 50%, more preferably >50%, most preferably >100% and that    blocks the bioactivity of ADM to less than 80%, preferably less than    50%.-   14. ADM antibody or an adrenomedullin antibody fragment or ADM    non-IG scaffold for use in therapy of a chronic or acute disease of    a patient according to any of the claims 1 to 13 to be used in    combination with catecholamine and/or fluids administered    intravenously.-   15. ADM antibody or adrenomedullin antibody fragment or ADM non-IG    scaffold for use in therapy of a chronic or acute disease of a    patient according to any of the claims 1 to 13 or a combination    according to claim 12 to be used in combination with ADM binding    protein and/or further active ingredients.-   16. Pharmaceutical formulation comprising an antibody or fragment or    scaffold according to any of claims 1 to 15.-   17. Pharmaceutical formulation according to claim 16 wherein said    pharmaceutical formulation is a solution, preferably a ready-to-use    solution.-   18. Pharmaceutical formulation according to claim 16 wherein said    pharmaceutical formulation is in a freeze-dried state.-   19. Pharmaceutical formulation according to any of claims 16 to 17,    wherein said pharmaceutical formulation is administered    intra-muscular.-   20. Pharmaceutical formulation according to any of claims 16 to 17,    wherein said pharmaceutical formulation is administered    intra-vascular.-   21. Pharmaceutical formulation according to claim 20, wherein said    pharmaceutical formulation is administered via infusion.

Further embodiments within the scope of the present invention are setout below:

-   1. Adrenomedullin (ADM) antibody or an adrenomedullin antibody    fragment for use in therapy of a chronic or acute disease of a    patient for stabilizing the circulation.-   2. ADM antibody or an adrenomedullin antibody fragment according to    claim 1 wherein said antibody or fragment reduces the catecholamine    requirement of said patient.-   3. ADM antibody or an adrenomedullin antibody fragment according to    claim 1 or 2 wherein the antibody format is selected from the group    comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment,    (Fab)2 fragment and scFv-Fc Fusion protein.-   4. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 3 wherein said antibody or fragment binds to the    N-terminal part (aa 1-21) of adrenomedullin.-   5. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 4, wherein said antibody or fragment recognizes    and binds to the N-terminal end (aa1) of adrenomedullin.-   6. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 5, wherein said antibody or fragment is an ADM    stabilizing antibody that enhances the t1/2 half retention time of    adrenomedullin in serum, blood, plasma at least 10%, preferably at    least, 50%, more preferably >50%, most preferably >100%.-   7. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 6, wherein said antibody or fragment blocks the    bioactivity of ADM to less than 80%, preferably less than 50%.-   8. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 7, wherein said antibody or fragment is a    modulating ADM antibody or a modulating adrenomedullin antibody    fragment that enhances the t1/2 half retention time of    adrenomedullin in serum, blood, plasma at least 10%, preferably at    least, 50%, more preferably >50%, most preferably >100% and that    blocks the bioactivity of ADM to less than 80%, preferably less than    50%:-   9. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronic or acute disease of a patient according to any    of the claims 1 to 8 wherein said disease is selected from the group    comprising sepsis, diabetis, cancer, acute and chronic vascular    diseases as e.g. heart failure, shock as e.g. septic shock and organ    dysfunction as e.g. kidney dysfunction.-   10. Pharmaceutical formulation comprising an antibody according to    any of claims 1 to 9.-   11. Pharmaceutical formulation according to claim 10 wherein said    pharmaceutical formulation is a solution, preferably a ready-to-use    solution.-   12. Pharmaceutical formulation according to claim 10 wherein said    pharmaceutical formulation is in a freeze-dried state.-   13. Pharmaceutical formulation according to any of claims 10 to 11,    wherein said pharmaceutical formulation is administered    intra-muscular.-   14. Pharmaceutical formulation according to any of claims 10 to 11,    wherein said pharmaceutical formulation is administered    intra-vascular.-   15. Pharmaceutical formulation according to claim 14, wherein said    pharmaceutical formulation is administered via infusion.

Further embodiments within the scope of the present invention are setout below:

-   1. Adrenomedullin (ADM) antibody or an adrenomedullin antibody    fragment or an ADM non-IG scaffold for use in therapy of a chronic    or acute disease or condition of a patient for stabilizing the    circulation.-   2. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to claim 1 wherein said antibody or fragment or    scaffold reduces the vasopressor requirement, e.g. catecholamine    requirement of said patient.-   3. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to claim 1 or 2 wherein said ADM antibody or an    adrenomedullin antibody fragment or ADM non-IG scaffold is a    non-neutralizing ADM antibody or a non-neutralizing adrenomedullin    antibody fragment or a non-neutralizing ADM non-IG scaffold.-   4. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 3 wherein the antibody format is selected from    the group comprising Fv fragment, scFv fragment, Fab fragment, scFab    fragment, (Fab)2 fragment and scFv-Fc Fusion protein.-   5. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 4 wherein said antibody or    fragment or scaffold binds to the N-terminal part (aa 1-21) of    adrenomedullin.-   6. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 5, wherein said antibody or    fragment or scaffold recognizes and binds to the N-terminal end    (aa1) of adrenomedullin.-   7. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 6, wherein said antibody or    fragment or scaffold is an ADM stabilizing antibody or fragment or    scaffold that enhances the half life (t1/2 half retention time) of    adrenomedullin in serum, blood, plasma at least 10%, preferably at    least, 50%, more preferably >50%, most preferably >100%.-   8. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 7, wherein said antibody or    fragment or scaffold blocks the bioactivity of ADM to less than 80%,    preferably less than 50%.-   9. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 8, wherein said antibody or    fragment or scaffold is a modulating ADM antibody or a modulating    adrenomedullin antibody fragment or scaffold that enhances the half    life (t1/2 half retention time) of adrenomedullin in serum, blood,    plasma at least 10%, preferably at least, 50%, more preferably >50%,    most preferably >100% and that blocks the bioactivity of ADM to less    than 80%, preferably less than 50%:-   10. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 9, wherein said antibody or fragment is a human    monoclonal antibody or fragment that binds to ADM or an antibody    fragment thereof wherein the heavy chain comprises the sequences

SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY

-   -   and wherein the light chain comprises the sequences

SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS SEQ ID NO: 6 FQGSHIPYT.

-   11. A human monoclonal antibody or fragment that binds to ADM or an    antibody fragment thereof according to claim 10 wherein said    antibody or fragment comprises a sequence selected from the group    comprising:

SEQ ID NO: 7 (AM-VH-C)QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 8 (AM-VH1)QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 9 (AM-VH2-E40)QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 10 (AM-VH3-T26-E55)QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 11 (AM-VH4-T26-E40-E55)QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 12 (AM-VL-C)DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO: 13 (AM-VL1)DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO: 14 (AM-VL2-E40)DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

-   12. ADM antibody or an adrenomedullin antibody fragment or ADM    non-IG scaffold for use in therapy of a chronic or acute disease of    a patient according to any of the claims 1 to 11 wherein said    disease is selected from the group comprising SIRS, sepsis,    diabetis, cancer, acute and chronic vascular diseases as e.g. heart    failure, shock as e.g. septic shock and organ dysfunction as e.g.    kidney dysfunction.-   13. ADM antibody or an adrenomedullin antibody fragment or ADM    non-IG scaffold for use in therapy of a chronic or acute disease of    a patient according to any of the claims 1 to 12 to be used in    combination with catecholamine and/or fluids administered    intravenously.-   14. ADM antibody or adrenomedullin antibody fragment or ADM non-IG    scaffold for use in therapy of a chronic or acute disease of a    patient according to any of the claims 1 to 13 or a combination    according to claim 10 to be used in combination with ADM binding    protein and/or further active ingredients.-   15. Pharmaceutical formulation comprising an antibody or fragment or    non-IG scaffold according to any of claims 1 to 14.-   16. Pharmaceutical formulation according to claim 15 wherein said    pharmaceutical formulation is a solution, preferably a ready-to-use    solution.-   17. Pharmaceutical formulation according to claim 15 wherein said    pharmaceutical formulation is in a freeze-dried state.-   18. Pharmaceutical formulation according to any of claims 15 to 16,    wherein said pharmaceutical formulation is administered    intra-muscular.-   19. Pharmaceutical formulation according to any of claims 14 to 16,    wherein said pharmaceutical formulation is administered    intra-vascular.-   20. Pharmaceutical formulation according to claim 16, wherein said    pharmaceutical formulation is administered via infusion.

Further embodiments within the scope of the present invention are setout below:

-   1. Adrenomedullin antibody or an adrenomedullin antibody fragment    for use in a treatment of a chronic or acute disease wherein said    antibody or said fragment is an ADM stabilizing antibody or fragment    that enhances the t_(1/2) half retention time of adrenomedullin in    serum, blood, plasma at least 10%, preferably at least, 50%, more    preferably >50%, most preferably 100% and/or wherein said antibody    blocks the bioactivity of ADM to less than 80%, preferably to less    than 50%.-   2. Adrenomedullin antibody or an adrenomedullin antibody fragment    for use in a treatment of a chronic or acute disease wherein said    antibody or said fragment is a modulating ADM antibody or fragment    that enhances the t_(1/2) half retention time of adrenomedullin in    serum, blood, plasma at least 10%, preferably at least, 50%, more    preferably >50%, most preferably 100% and that blocks the    bioactivity of ADM to less than 80%, preferably to less than 50%.-   3. Adrenomedullin antibody or an adrenomedullin antibody fragment    for use in a treatment of a chronic or acute disease according to    claim 1 or 2, wherein said antibody or fragment binds to the    N-terminal part (aa 1-21) of adrenomedullin.-   4. Adrenomedullin antibody or an adrenomedullin antibody fragment    for use in a treatment of a chronic or acute disease wherein said    antibody or said fragment according to claim 3 binds to the    N-terminal end of adrenomedullin.-   5. Adrenomedullin antibody or an adrenomedullin antibody fragment    for use in use in a treatment of a chronic or acute disease    according to any of claims 1 to 4, wherein said antibody or said    fragment is an ADM stabilizing antibody or fragment that enhances    the t₁₁₂ half retention time of adrenomedullin in serum, blood,    plasma at least 10%, preferably at least, 50%, more preferably >50%,    most preferably 100%.-   6. Adrenomedullin antibody or an adrenomedullin antibody fragment    for use in a treatment of a chronic or acute disease according to    any of claims 1 to 5, wherein said antibody or said fragment blocks    the bioactivity of ADM to less than 80%, preferably to less than    50%.-   7. Adrenomedullin antibody or an adrenomedullin antibody fragment    according to any of the claims 1 to 6 for use in a treatment of a    chronic or acute disease wherein said disease is selected from the    group comprising SIRS, sepsis, septic shock, diabetis, cancer, heart    failure, shock, organ failure, kidney dysfunction, acute liquid    dysbalance, and low blood pressure.-   8. Adrenomedullin antibody or an adrenomedullin antibody fragment    according to any of the claims 1 to 7 for use in a treatment of a    chronic or acute disease wherein said disease is septic shock or    sepsis.-   9. Adrenomedullin antibody or an adrenomedullin antibody fragment    for use in a treatment of a chronic or acute disease according to    any of the claims 1 to 8 wherein said antibody or fragment regulates    the liquid balance of said patient.-   10. Adrenomedullin antibody or an adrenomedullin antibody fragment    for use in a treatment of a chronic or acute disease according to    any of the claims 1 to 9 wherein said antibody or fragment used for    prevention of organ dysfunction or organ failure.-   11. Adrenomedullin antibody or an adrenomedullin antibody fragment    for use in a treatment of a chronic or acute disease according to    claim 10 wherein said antibody or fragment is used for prevention of    kidney dysfunction or kidney failure.-   12. Adrenomedullin (ADM) antibody or an adrenomedullin antibody    fragment for use in a treatment of a chronic or acute disease in a    patient according to claims 1 to 11 wherein said antibody or    fragment is used for stabilizing the circulation.-   13. ADM antibody or an adrenomedullin antibody fragment for use in a    treatment of a chronic or acute disease in a patient according to    claim 12 wherein said antibody or fragment reduces the catecholamine    requirement of said patient.-   14. ADM antibody or an adrenomedullin antibody fragment for use in a    treatment of a chronic or acute disease in a patient according to    any of claims 1 to 13 for the reduction of the mortality risk for    said patient.-   15. ADM antibody or an adrenomedullin antibody fragment for use in a    treatment of a chronic or acute disease in a patient according to    any of claims 1 to 14 wherein said antibody or fragment may be    administered in a dose of at least 3 μg/Kg body weight.-   16. Pharmaceutical composition comprising an antibody or fragment    according to any of claims 1 to 15.

Further embodiments within the scope of the present invention are setout below:

-   1. Adrenomedullin antibody or an adrenomedullin antibody fragment or    ADM non-Ig scaffold wherein said antibody or said fragment or    scaffold is a non-neutralizing antibody.-   2. Adrenomedullin antibody or an adrenomedullin antibody fragment or    ADM non-Ig scaffold wherein said antibody or said fragment or    scaffold is an ADM stabilizing antibody or fragment or scaffold that    enhances the half life (t_(1/2) half retention time) of    adrenomedullin in serum, blood, plasma at least 10%, preferably at    least 50%, more preferably >50%, most preferably 100% and/or wherein    said antibody or fragment or scaffold blocks the bioactivity of ADM    to less than 80%, preferably to less than 50%.-   3. Adrenomedullin antibody or an adrenomedullin antibody fragment or    ADM non-Ig scaffold wherein said antibody or said fragment is a    modulating ADM antibody or fragment or scaffold that enhances the    half life (t_(1/2) half retention time) of adrenomedullin in serum,    blood, plasma at least 10%, preferably at least, 50%, more    preferably >50%, most preferably 100% and that blocks the    bioactivity of ADM to less than 80%, preferably to less than 50%.-   4. Adrenomedullin antibody or an adrenomedullin antibody fragment or    ADM non-Ig scaffold according to claim 1 or 2, wherein said antibody    or fragment or scaffold binds to the N-terminal part (aa 1-21) of    adrenomedullin.-   5. Adrenomedullin antibody or an adrenomedullin antibody fragment or    ADM non-Ig scaffold wherein said antibody or said fragment or    scaffold according to claim 3 binds to the N-terminal end of    adrenomedullin.-   6. Adrenomedullin antibody or an adrenomedullin antibody fragment    ADM non-Ig scaffold according to any of claims 1 to 4, wherein said    antibody or said fragment or said scaffold is an ADM stabilizing    antibody or fragment that enhances the t_(1/2) half retention time    of adrenomedullin in serum, blood, plasma at least 10%, preferably    at least, 50%, more preferably >50%, most preferably 100%.-   7. Adrenomedullin antibody or an adrenomedullin antibody fragment or    ADM non-Ig scaffold according to any of the claims 1 to 6 for use as    an active pharmaceutical substance.-   8. Adrenomedullin antibody or an adrenomedullin antibody fragment    ADM non-Ig scaffold according to any of the claims 1 to 7 for use in    a treatment of a chronic or acute disease or acute condition wherein    said disease or condition is selected from the group comprising    severe infections as e.g. meningitis, systemic inflammatory    Response-Syndrome (SIRS,) sepsis; other diseases as diabetes,    cancer, acute and chronic vascular diseases as e.g. heart failure,    myocardial infarction, stroke, atherosclerosis; shock as e.g. septic    shock and organ dysfunction as e.g. kidney dysfunction, liver    dysfunction, burnings, surgery, traumata.-   9. Adrenomedullin antibody or an adrenomedullin antibody fragment or    ADM non-Ig scaffold according to any of the claims 1 to 8 for use in    a treatment of a chronic or acute disease or acute condition wherein    said disease is septic shock or sepsis.-   10. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 9, wherein said antibody or fragment is a human    monoclonal antibody or fragment that binds to ADM or an antibody    fragment thereof wherein the heavy chain comprises at least one of    the sequences:

SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY

-   -   And/or wherein the light chain comprises the at least one of the        sequences

SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS SEQ ID NO: 6 FQGSHIPYT.

-   11. A human monoclonal antibody or fragment that binds to ADM or an    antibody fragment thereof according to claim 10 wherein said    antibody or fragment comprises a sequence selected from the group    comprising:

SEQ ID NO: 7 (AM-VH-C)QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 8 (AM-VH1)QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 9 (AM-VH2-E40)QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 10 (AM-VH3-T26-E55)QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 11 (AM-VH4-T26-E40-E55)QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 12 (AM-VL-C)DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO: 13 (AM-VL1)DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO: 14 (AM-VL2-E40)DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

-   12. Adrenomedullin antibody or an adrenomedullin antibody fragment    or ADM non-Ig scaffold according to any of the claims 1 to 11 for    regulating the fluid balance in a patient having a chronic or acute    disease or acute condition.-   13. Adrenomedullin antibody or an adrenomedullin antibody fragment    or ADM non-Ig scaffold according to any of the claims 1 to 11 for    preventing or reducing organ dysfunction or organ failure in a    patient having in a chronic or acute disease or acute condition.-   14. Adrenomedullin antibody or an adrenomedullin antibody fragment    or ADM non-Ig scaffold according to claim 10 wherein organ is kidney    or liver.-   15. Adrenomedullin (ADM) antibody or an adrenomedullin antibody    fragment or ADM non-Ig scaffold according to claims 1 to 14 for    stabilizing the circulation in a patient having a chronic or acute    disease or acute condition.-   16. ADM antibody or an adrenomedullin antibody fragment or ADM    non-Ig scaffold for use in a treatment of a chronic or acute disease    in a patient according to claim 15 wherein said antibody or fragment    reduces the catecholamine requirement of said patient.-   17. Adrenomedullin antibody or an adrenomedullin antibody fragment    or ADM non-Ig scaffold according to any of the claims 1 to 16 to be    used in combination with vasopressors e.g. catecholamine.-   18. Adrenomedullin antibody or an adrenomedullin antibody fragment    or ADM non-Ig scaffold according to any of the claims 1 to 17 to be    used in combination with intravenous fluid administration.-   19. Adrenomedullin antibody or an adrenomedullin antibody fragment    or ADM non-Ig scaffold according to any of the claims 1 to 18 to be    used in combination with an TNF-alpha-antibody.-   20. ADM antibody or an adrenomedullin antibody fragment or    non-Ig-scaffold according to any of claims 1 to 19 for use in a    treatment of a patient in need thereof wherein said antibody or    fragment may be administered in a dose of at least 3 μg/Kg body    weight.-   21. Pharmaceutical composition comprising an antibody or fragment or    scaffold according to any of claims 1 to 20.-   22. ADM antibody or an adrenomedullin antibody fragment or    non-Ig-scaffold according to any of claims 1 to 20 for use in a    treatment of a chronic or acute disease or chronic condition.-   23. ADM antibody or an adrenomedullin antibody fragment or    non-Ig-scaffold according to claim 22 wherein said disease is    sepsis.

Further embodiments within the scope of the present invention are setout below:

-   1. Adrenomedullin ADM antibody or an adrenomedullin antibody    fragment for use in therapy of a severe chronical or acute disease    of a patient for the reduction of the mortality risk for said    patient.-   2. ADM antibody or an adrenomedullin antibody fragment according to    claim 1 wherein the antibody format is selected from the group    comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment,    (Fab)2 fragment and scFv-Fc Fusion protein.-   3. ADM antibody or an adrenomedullin antibody fragment according    claim 1 or 2 wherein said antibody or fragment binds to the    N-terminal part (aa 1-21) of adrenomedullin.-   4. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 3, wherein said antibody or fragment recognizes    and binds to the N-terminal end (aa1) of adrenomedullin.-   5. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 4, wherein said antibody or fragment is an ADM    stabilizing antibody or fragment that enhances the t1/2 half    retention time of adrenomedullin in serum, blood, plasma at least    10%, preferably at least, 50%, more preferably >50%, most preferably    >100%.-   6. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 5, wherein said antibody or fragment blocks the    bioactivity of ADM to less than 80%, preferably less than 50%.-   7. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronical or acute disease of a patient according to    any of claims 1 to 6 wherein said disease is selected from the group    comprising sepsis, diabetis, cancer, heart failure, shock and kidney    dysfunction.-   8. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronical or acute disease of a patient according to    any of claims 1 to 7 wherein said patient is an ICU patient.-   9. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronical or acute disease of a patient according to    any of claims 1 to 8 wherein the mortality risk is reduced by    preventing adverse event wherein the latter are selected from the    group comprising SIRS, sepsis, septic shock, organ failure, kidney    failure, liquid dysbalance and low blood pressure.-   10. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronical or acute disease of a patient according to    any of claims 1 to 8 wherein said antibody or fragment is to be used    in combination of ADM binding protein.-   11. Pharmaceutical formulation comprising an antibody or fragment    according to any of claims 1 to 10.-   12. Pharmaceutical formulation according to claim 11 wherein said    pharmaceutical formulation is a solution, preferably a ready-to-use    solution.-   13. Pharmaceutical formulation according to claim 11 wherein said    pharmaceutical formulation is in a freeze-dried state.-   14. Pharmaceutical formulation according to any of claims 11 to 12,    wherein said pharmaceutical formulation is administered    intra-muscular.-   15. Pharmaceutical formulation according to any of claims 11 to 12,    wherein said pharmaceutical formulation is administered    intra-vascular.-   16. Pharmaceutical formulation according to claim 15, wherein said    pharmaceutical formulation is administered via infusion.

Further embodiments within the scope of the present invention are setout below:

-   1. Adrenomedullin (ADM) antibody or an adrenomedullin antibody    fragment or ADM non-Ig scaffold for use in therapy of a severe    chronical or acute disease or acute condition of a patient for the    reduction of the mortality risk for said patient wherein said    antibody or fragment or scaffold is a non-neutralizing ADM antibody    or a non-neutralizing adrenomedullin antibody fragment or a    non-neutralizing ADM non-Ig scaffold.-   2. ADM antibody or an adrenomedullin antibody fragment according to    claim 1 wherein the antibody format is selected from the group    comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment,    (Fab)2 fragment and scFv-Fc Fusion protein.-   3. ADM antibody or an adrenomedullin antibody fragment or an ADM    non-Ig scaffold according claim 1 or 2 wherein said antibody or    fragment or scaffold binds to the N-terminal part (aa 1-21) of    adrenomedullin.-   4. ADM antibody or an adrenomedullin antibody fragment or an ADM    non-Ig scaffold according to any of claims 1 to 3, wherein said    antibody or fragment or scaffold recognizes and binds to the    N-terminal end (aa1) of adrenomedullin.-   5. ADM antibody or an adrenomedullin antibody fragment or an ADM    non-Ig scaffold according to any of claims 1 to 4, wherein said    antibody or fragment or scaffold is an ADM stabilizing antibody or    fragment or scaffold that enhances the half life (t1/2 half    retention time) of adrenomedullin in serum, blood, plasma at least    10%, preferably at least, 50%, more preferably >50%, most preferably    >100%.-   6. ADM antibody or an adrenomedullin antibody fragment or an ADM    non-Ig scaffold according to any of claims 1 to 5, wherein said    antibody or fragment or scaffold blocks the bioactivity of ADM to    less than 80%, preferably less than 50%.-   7. ADM antibody or an adrenomedullin antibody fragment or an ADM    non-Ig scaffold for use in therapy of a chronical or acute disease    of a patient according to any of claims 1 to 6 wherein said disease    is selected from the group comprising severe infections as e.g.    meningitis, Systemic inflammatory Response-Syndrom (SIRS,) sepsis;    other diseases as diabetis, cancer, acute and chronic vascular    diseases as e.g. heart failure, myocardial infarction, stroke,    atherosclerosis; shock as e.g. septic shock and organ dysfunction as    e.g. kidney dysfunction, liver dysfunction; burnings, surgery,    traumata.-   8. ADM antibody or an adrenomedullin antibody fragment or an ADM    non-Ig scaffold for use in therapy of a chronical or acute disease    of a patient according to any of claims 1 to 7 wherein said disease    is selected from the group comprising SIRS, a severe infection,    sepsis, shock e.g. septic shock.-   9. ADM antibody or an adrenomedullin antibody fragment or an ADM    non-Ig scaffold for use in therapy of a chronical or acute disease    or acute condition of a patient according to any of claims 1 to 8    wherein said patient is an ICU patient. ADM antibody or an    adrenomedullin antibody fragment or an ADM non-Ig scaffold for use    in therapy of a chronical or acute disease or acute condition of a    patient according to any of claims 1 to 9 wherein the mortality risk    is reduced by preventing an adverse event wherein the latter are    selected from the group comprising SIRS, sepsis, shock as e.g.    septic shock, acute and chronic vascular diseases as e.g. acute    heart failure, myocardial infarction, stroke; organ failure as e.g,    kidney failure, liver failure, fluid dysbalance and low blood    pressure.-   10. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 9, wherein said antibody or fragment is a human    monoclonal antibody or fragment that binds to ADM or an antibody    fragment thereof wherein the heavy chain comprises the sequences

SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY

-   -   and wherein the light chain comprises the sequences

SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS SEQ ID NO: 6 FQGSHIPYT.

-   12. A human monoclonal antibody or fragment that binds to ADM or an    antibody fragment thereof according to claim 10 wherein said    antibody or fragment comprises a sequence selected from the group    comprising:

SEQ ID NO: 7 (AM-VH-C)QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 8 (AM-VH1)QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 9 (AM-VH2-E40)QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 10 (AM-VH3-T26-E55)QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 11 (AM-VH4-T26-E40-E55)QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 12 (AM-VL-C)DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO: 13 (AM-VL1)DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO: 14 (AM-VL2-E40)DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

-   13. ADM antibody or an adrenomedullin antibody fragment or ADM    non-IG scaffold for use in therapy of a chronic or acute disease of    a patient according to any of the claims 1 to 12 to be used in    combination with vasopressors e.g. catecholamine and/or fluids    administered intravenously.-   14. ADM antibody or adrenomedullin antibody fragment or ADM non-IG    scaffold for use in therapy of a chronic or acute disease of a    patient according to any of the claims 1 to 13 or a combination    according to claim 10 to be used in combination with ADM binding    protein and/or further active ingredients.-   15. Pharmaceutical formulation comprising an antibody or fragment or    scaffold according to any of claims 1 to 14.-   16. Pharmaceutical formulation according to claim 15 wherein said    pharmaceutical formulation is a solution, preferably a ready-to-use    solution.-   17. Pharmaceutical formulation according to claim 15 wherein said    pharmaceutical formulation is in a freeze-dried state.-   18. Pharmaceutical formulation according to any of claims 15 to 16,    wherein said pharmaceutical formulation is administered    intra-muscular.-   19. Pharmaceutical formulation according to any of claims 15 to 16,    wherein said pharmaceutical formulation is administered    intra-vascular.-   20. Pharmaceutical formulation according to claim 19, wherein said    pharmaceutical formulation is administered via infusion.-   21. ADM antibody or an Adrenomedullin antibody fragment or AM non-Ig    scaffold, wherein said antibody or fragment or scaffold binds to the    N-terminal part (aa 1-21) of Adrenomedullin in, preferably human    ADM.-   22. Antibody or fragment or scaffold according to claim 2, wherein    said antibody or fragment or scaffold recognizes and binds to the    N-terminal end (aa 1) of Adrenomedullin.

Further embodiments within the scope of the present invention are setout below:

-   1. Adrenomedullin (ADM) antibody or an adrenomedullin antibody    fragment for use in therapy of a chronical or acute disease of a    patient for prevention of organ dysfunction or organ failure.-   2. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronical or acute disease according to claim 1 wherein    said organ is kidney.-   3. ADM antibody or an adrenomedullin antibody fragment according to    claim 1 wherein the antibody format is selected from the group    comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment,    (Fab)2 fragment and scFv-Fc Fusion protein.-   4. ADM antibody or an adrenomedullin antibody fragment according any    of claims 1 to 3 wherein said antibody or fragment binds to the    N-terminal part (aa 1-21) of adrenomedullin.-   5. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 4, wherein said antibody or fragment recognizes    and binds to the N-terminal end (aa1) of adrenomedullin.-   6. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 5, wherein said antibody or said fragment is an    ADM stabilizing antibody or fragment that enhances the t1/2 half    retention time of adrenomedullin in serum, blood, plasma at least    10%, preferably at least 50%, more preferably >50%, most preferably    >100%.-   7. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 6, wherein said antibody blocks the bioactivity    of ADM to less than 80%, preferably less than 50%.-   8. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronical or acute disease of a patient according to    any of claims 1 to 7 wherein said disease is selected from the group    comprising sepsis, diabetis, cancer, heart failure, and shock.-   9. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronical or acute disease of a patient according to    any of claims 1 to 8 wherein said patient is an ICU patient.-   10. ADM antibody or an adrenomedullin antibody fragment for use in    therapy of a chronical or acute disease of a patient according to    any of claims 1 to 9 wherein said antibody or fragment is a    modulating antibody or fragment that enhances the t1/2 half    retention time of adrenomedullin in serum, blood, plasma at least    10%, preferably at least 50%, more preferably >50%, most    preferably >100% and that blocks the bioactivity of ADM to less than    80%, preferably less than 50%.-   11. Pharmaceutical formulation comprising an antibody or fragment    according to any of claims 1 to 10.-   12. Pharmaceutical formulation according to claim 11 wherein said    pharmaceutical formulation is a solution, preferably a ready-to-use    solution.-   13. Pharmaceutical formulation according to claim 11 wherein said    pharmaceutical formulation is in a freeze-dried state.-   14. Pharmaceutical formulation according to any of claims 11 to 12,    wherein said pharmaceutical formulation is administered    intra-muscular.-   15. Pharmaceutical formulation according to any of claims 11 to 12,    wherein said pharmaceutical formulation is administered    intra-vascular.-   16. Pharmaceutical formulation according to claim 15, wherein said    pharmaceutical formulation is administered via infusion.

Further embodiments within the scope of the present invention are setout below:

-   1. Adrenomedullin (ADM) antibody or an adrenomedullin antibody    fragment or ADM non-Ig scaffold for use in therapy of a chronical or    acute disease or acute condition of a patient for prevention or    reduction of organ dysfunction or prevention of organ failure in    said patient.-   2. ADM antibody or an adrenomedullin antibody fragment or ADM non-Ig    scaffold for use in therapy of a chronical or acute disease or acute    disease according to claim 1 wherein said organ is kidney or liver.-   3. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to claim 1 or 2 wherein said ADM antibody or an    adrenomedullin antibody fragment or ADM non-IG scaffold is a    non-neutralizing ADM antibody or a non-neutralizing adrenomedullin    antibody fragment or a non-neutralizing ADM non-IG scaffold-   4. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claim 1 or 3 wherein the antibody    format is selected from the group comprising Fv fragment, scFv    fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFv-Fc    Fusion protein.-   5. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according any of claims 1 to 4 wherein said antibody or    fragment or scaffold binds to the N-terminal part (aa 1-21) of    adrenomedullin.-   6. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 5, wherein said antibody or    fragment or scaffold recognizes and binds to the N-terminal end    (aa1) of adrenomedullin.-   7. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 6, wherein said antibody or    said fragment or scaffold is an ADM stabilizing antibody or fragment    or scaffold that enhances the half life (t1/2 half retention time)    of adrenomedullin in serum, blood, plasma at least 10%, preferably    at least 50%, more preferably >50%, most preferably >100%.-   8. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold according to any of claims 1 to 7, wherein said antibody or    fragment or scaffold blocks the bioactivity of ADM to less than 80%,    preferably less than 50%.-   9. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG    scaffold for use in therapy of a chronical or acute disease or acute    condition of a patient according to any of claims 1 to 8 wherein    said disease is selected from the group comprising sepsis, diabetis,    cancer, heart failure, and shock.-   10. ADM antibody or an adrenomedullin antibody fragment according to    any of claims 1 to 9, wherein said antibody or fragment is a human    monoclonal antibody or fragment that binds to ADM or an antibody    fragment thereof wherein the heavy chain comprises the sequences

SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY

-   -   and wherein the light chain comprises the sequences

SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS SEQ ID NO: 6 FQGSHIPYT.

-   11. A human monoclonal antibody or fragment that binds to ADM or an    antibody fragment thereof according to claim 10 wherein said    antibody or fragment comprises a sequence selected from the group    comprising:

SEQ ID NO: 7 (AM-VH-C)QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 8 (AM-VH1)QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 9 (AM-VH2-E40)QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 10 (AM-VH3-T26-E55)QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 11 (AM-VH4-T26-E40-E55)QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 12 (AM-VL-C)DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO: 13 (AM-VL1)DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECSEQ ID NO: 14 (AM-VL2-E40)DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

-   12. ADM antibody or an adrenomedullin antibody fragment or ADM    non-IG scaffold for use in therapy of a chronical or acute disease    of a patient according to any of claims 1 to 11 wherein said    antibody or fragment or scaffold is a modulating antibody or    fragment or scaffold that enhances the half life (t1/2 half    retention time) of adrenomedullin in serum, blood, plasma at least    10%, preferably at least 50%, more preferably >50%, most    preferably >100% and that blocks the bioactivity of ADM to less than    80%, preferably less than 50%.-   13. ADM antibody or an adrenomedullin antibody fragment or ADM    non-IG scaffold for use in therapy of a chronic or acute disease or    acute condition of a patient according to any of the claims 1 to 12    to be used in combination with vasopressors e.g. catecholamine    and/or fluids administered intravenously.-   14. ADM antibody or adrenomedullin antibody fragment or ADM non-IG    scaffold for use in therapy of a chronic or acute disease or acute    condition of a patient according to any of the claims 1 to 13 or a    combination according to claim 13 to be used in combination with ADM    binding protein and/or further active ingredients.-   15. Pharmaceutical formulation comprising an antibody or fragment    according to any of claims 1 to 13.-   16. Pharmaceutical formulation according to claim 14 wherein said    pharmaceutical formulation is a solution, preferably a ready-to-use    solution.-   17. Pharmaceutical formulation according to claim 14 wherein said    pharmaceutical formulation is in a freeze-dried state.-   18. Pharmaceutical formulation according to any of claims 14 to 15,    wherein said pharmaceutical formulation is administered    intra-muscular.-   19. Pharmaceutical formulation according to any of claims 14 to 15,    wherein said pharmaceutical formulation is administered    intra-vascular.-   20. Pharmaceutical formulation according to claim 18, wherein said    pharmaceutical formulation is administered via infusion.

EXAMPLES

It should be emphasized that the antibodies, antibody fragments andnon-Ig scaffolds of the example portion in accordance with the inventionare binding to ADM, and thus should be considered as anti-ADMantibodies/antibody fragments/non-Ig scaffolds.

Example 1 Generation of Antibodies and Determination of their AffinityConstants

Several human and murine antibodies were produced and their affinityconstants were determined (see tables 1 and 2).

Peptides/Conjugates for Immunization:

Peptides for immunization were synthesized, see Table 1, OPTTechnologies, Berlin, Germany) with an additional N-terminal Cystein (ifno Cystein is present within the selected ADM-sequence) residue forconjugation of the peptides to Bovine Serum Albumin (BSA). The peptideswere covalently linked to BSA by using Sulfolink-coupling gel(Perbio-science, Bonn, Germany). The coupling procedure was performedaccording to the manual of Perbio.

The murine antibodies were generated according to the following method:

A Balb/c mouse was immunized with 100 μg Peptide-BSA-Conjugate at day 0and 14 (emulsified in 100 μl complete Freund's adjuvant) and 50 μg atday 21 and 28 (in 100 μl incomplete Freund's adjuvant). Three daysbefore the fusion experiment was performed, the animal received 50 μg ofthe conjugate dissolved in 100 μl saline, given as one intraperitonealand one intra-venous injection.

Spenocytes from the immunized mouse and cells of the myeloma cell lineSP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C.After washing, the cells were seeded in 96-well cell culture plates.Hybrid clones were selected by growing in HAT medium [RPMI 1640 culturemedium supplemented with 20% fetal calf serum and HAT-Supplement]. Aftertwo weeks the HAT medium is replaced with HT Medium for three passagesfollowed by returning to the normal cell culture medium.

The cell culture supernatants were primary screened for antigen specificIgG antibodies three weeks after fusion. The positive testedmicrocultures were transferred into 24-well plates for propagation.After retesting, the selected cultures were cloned and recloned usingthe limiting-dilution technique and the isotypes were determined.

(see also Lane, R. D. “A short-duration polyethylene glycol fusiontechnique for increasing production of monoclonal antibody-secretinghybridomas”, J. Immunol. Meth. 81: 223-228; (1985), Ziegler, B. et al.“Glutamate decarboxylase (GAD) is not detectable on the surface of ratislet cells examined by cytofluorometry and complement-dependentantibody-mediated cytotoxicity of monoclonal GAD antibodies”, Horm.Metab. Res. 28: 11-15, (1996)).

Mouse Monoclonal Antibody Production:

Antibodies were produced via standard antibody production methods (Marxet al, Monoclonal Antibody Production, ATLA 25, 121, 1997,) and purifiedvia Protein A. The antibody purities were >95% based on SDS gelelectrophoresis analysis.

Human Antibodies

Human Antibodies were produced by means of phage display according tothe following procedure:

The human naive antibody gene libraries HALT/8 were used for theisolation of recombinant single chain F-Variable domains (scFv) againstadrenomedullin peptide. The antibody gene libraries were screened with apanning strategy comprising the use of peptides containing a biotin taglinked via two different spacers to the adrenomedullin peptide sequence.A mix of panning rounds using non-specifically bound antigen andstreptavidin bound antigen were used to minimize background ofnon-specific binders. The eluted phages from the third round of panninghave been used for the generation of monoclonal scFv expressing E. colistrains. Supernatant from the cultivation of these clonal strains hasbeen directly used for an antigen ELISA testing (see also Hust, M.,Meyer, T., Voedisch, B., Rülker, T., Thie, H., El-Ghezal, A., Kirsch, M.I., Schütte, M., Helmsing, S., Meier, D., Schirrmann, T., Dübel, S.,2011. A human scFv antibody generation pipeline for proteome research.Journal of Biotechnology 152, 159-170; Schütte, M., Thullier, P., Pelat,T., Wezler, X., Rosenstock, P., Hinz, D., Kirsch, M. I., Hasenberg, M.,Frank, R., Schirrmann, T., Gunzer, M., Hust, M., Dübel, S., 2009.Identification of a putative Crf splice variant and generation ofrecombinant antibodies for the specific detection of Aspergillusfumigatus. PLoS One 4, e6625).

Positive clones have been selected based on positive ELISA signal forantigen and negative for streptavidin coated micro titer plates. Forfurther characterizations the scFv open reading frame has been clonedinto the expression plasmid pOPE107 (Hust et al., J. Biotechn. 2011),captured from the culture supernatant via immobilised metal ion affinitychromatography and purified by a size exclusion chromatography.

Affinity Constants

To determine the affinity of the antibodies to Adrenomedullin, thekinetics of binding of Adrenomedullin to immobilized antibody wasdetermined by means of label-free surface plasmon resonance using aBiacore 2000 system (GE Healthcare Europe GmbH, Freiburg, Germany).Reversible immobilization of the antibodies was performed using ananti-mouse Fc antibody covalently coupled in high density to a CM5sensor surface according to the manufacturer's instructions (mouseantibody capture kit; GE Healthcare). (Lorenz et al., “FunctionalAntibodies Targeting IsaA of Staphylococcus aureus Augment Host ImmuneResponse and Open New Perspectives for Antibacterial Therapy”;Antimicrob Agents Chemother. 2011 January; 55(1): 165-173.)

The monoclonal antibodies were raised against the below depicted ADMregions of human and murine ADM, respectively. The following tablerepresents a selection of obtained antibodies used in furtherexperiments. Selection was based on target region:

TABLE 1 Affinity Sequence ADM constants Number Antigen/Immunogen RegionDesignation Kd (M) SEQ ID: 15 YRQSMNNFQGLRSFGCRFGTC  1-21 NT-H 5.9 ×10⁻⁹ SEQ ID: 16 CTVQKLAHQIYQ 21-32 MR-H   2 × 10⁻⁹ SEQ ID: 17CAPRSKISPQGY-NH2  C-42- CT-H 1.1 × 10⁻⁹ 52 SEQ ID: 18YRQSMNQGSRSNGCRFGTC  1-19 NT-M 3.9 × 10⁻⁹ SEQ ID: 19 CTFQKLAHQIYQ 19-31MR-M 4.5 × 10⁻¹⁰ SEQ ID: 20 CAPRNKISPQGY-NH2  C-40- CT-M   9 × 10⁻⁹ 50

The Following is a List of Further Obtained Monoclonal Antibodies:

List of Anti-ADM-Antibodies

TABLE 2 max inhibition Affinity bioassay (%) Target Source Klone number(M) (see example 2) NT-M Mouse ADM/63 5.8 × 10⁻⁹ 45 Mouse ADM/364 2.2 ×10⁻⁸ 48 Mouse ADM/365 3.0 × 10⁻⁸ Mouse ADM/366 1.7 × 10⁻⁸ Mouse ADM/3671.3 × 10⁻⁸ Mouse ADM/368 1.9 × 10⁻⁸ Mouse ADM/369 2.0 × 10⁻⁸ MouseADM/370 1.6 × 10⁻⁸ Mouse ADM/371 2.0 × 10⁻⁸ Mouse ADM/372 2.5 × 10⁻⁸Mouse ADM/373 1.8 × 10⁻⁸ Mouse ADM/377 1.5 × 10⁻⁸ Mouse ADM/378 2.2 ×10⁻⁸ Mouse ADM/379 1.6 × 10⁻⁸ Mouse ADM/380 1.8 × 10⁻⁸ Mouse ADM/381 2.4× 10⁻⁸ Mouse ADM/382 1.6 × 10⁻⁸ Mouse ADM/383 1.8 × 10⁻⁸ Mouse ADM/3841.7 × 10⁻⁸ Mouse ADM/385 1.7 × 10⁻⁸ Mouse ADM/403 1.2 × 10⁻⁸ MouseADM/395 1.2 × 10⁻⁸ Mouse ADM/396 3.0 × 10⁻⁸ Mouse ADM/397 1.5 × 10⁻⁸MR-M Mouse ADM/38 4.5 × 10⁻¹⁰ 68 MR-M Mouse ADM/39 5.9 × 10⁻⁹ 72 CT-MMouse ADM/65 9.0 × 10⁻⁹ 100 CT-M Mouse ADM/66 1.6 × 10⁻⁸ 100 NT-H MouseADM/33 5.9 × 10⁻⁸ 38 NT-H Mouse ADM/34 1.6 × 10⁻⁸ 22 MR-H Mouse ADM/411.2 × 10⁻⁸ 67 MR-H Mouse ADM/42  <1 × 10⁻⁸ MR-H Mouse ADM/43 2.0 × 10⁻⁹73 MR-H Mouse ADM/44  <1 × 10⁻⁸ CT-H Mouse ADM/15  <1 × 10⁻⁸ CT-H MouseADM/16 1.1 × 10⁻⁹ 100 CT-H Mouse ADM/17 3.7 × 10⁻⁹ 100 CT-H Mouse ADM/18 <1 × 10⁻⁸ hADM Phage display ADM/A7  <1 × 10⁻⁸ Phage display ADM/B7  <1× 10⁻⁸ Phage display ADM/C7  <1 × 10⁻⁸ Phage display ADM/G3  <1 × 10⁻⁸Phage display ADM/B6  <1 × 10⁻⁸ Phage display ADM/B11  <1 × 10⁻⁸ Phagedisplay ADM/D8  <1 × 10⁻⁸ Phage display ADM/D11  <1 × 10⁻⁸ Phage displayADM/G12  <1 × 10⁻⁸

Generation of Antibody Fragments by Enzymatic Digestion:

The generation of Fab and F(ab)₂ fragments was done by enzymaticdigestion of the murine full length antibody NT-M. Antibody NT-M wasdigested using a) the pepsin-based F(ab)₂ Preparation Kit (Pierce 44988)and b) the papain-based Fab Preparation Kit (Pierce 44985). Thefragmentation procedures were performed according to the instructionsprovided by the supplier. Digestion was carried out in case ofF(ab)₂-fragmentation for 8 h at 37° C. The Fab-fragmentation digestionwas carried out for 16 h, respectively.

Procedure for Fab Generation and Purification:

The immobilized papain was equilibrated by washing the resin with 0.5 mlof Digestion Buffer and centrifuging the column at 5000×g for 1 minute.The buffer was discarded afterwards. The desalting column was preparedby removing the storage solution and washing it with digestion buffer,centrifuging it each time afterwards at 1000×g for 2 minutes. 0.5 ml ofthe prepared IgG sample where added to the spin column tube containingthe equilibrated Immobilized Papain. Incubation time of the digestionreaction was done for 16 h on a tabletop rocker at 37° C. The column wascentrifuged at 5000×g for 1 minute to separate digest from theImmobilized Papain. Afterwards the resin was washed with 0.5 ml PBS andcentrifuged at 5000×g for 1 minute. The wash fraction was added to thedigested antibody that the total sample volume was 1.0 ml. The NAbProtein A Column was equilibrated with PBS and IgG Elution Buffer atroom temperature. The column was centrifuged for 1 minute to removestorage solution (contains 0.02% sodium azide) and equilibrated byadding 2 ml of PBS, centrifuge again for 1 minute and the flow-throughdiscarded. The sample was applied to the column and resuspended byinversion. Incubation was done at room temperature with end-over-endmixing for 10 minutes. The column was centrifuged for 1 minute, savingthe flow-through with the Fab fragments.

(References: Coulter, A. and Harris, R. (1983). J. Immunol. Meth. 59,199-203.; Lindner I. et al. (2010) { alpha}2-Macroglobulin inhibits themalignant properties of astrocytoma cells by impeding { beta}-cateninsignaling. Cancer Res. 70, 277-87.; Kaufmann B. et al. (2010)Neutralization of West Nile virus by cross-linking of its surfaceproteins with Fab fragments of the human monoclonal antibody CR4354.PNAS. 107, 18950-5.; Chen X. et al. (2010) Requirement of open headpiececonformation for activation of leukocyte integrin ax132. PNAS. 107,14727-32.; Uysal H. et al. (2009) Structure and pathogenicity ofantibodies specific for citrullinated collagen type II in experimentalarthitis. J. Exp. Med. 206, 449-62.; Thomas G. M. et al. (2009) Cancercell-derived microparticles bearing P-selectin glycoprotein ligand 1accelerate thrombus formation in vivo. J. Exp. Med. 206, 1913-27.; KongF. et al. (2009) Demonstration of catch bonds between an integrin andits ligand. J. Cell Biol. 185, 1275-84.)

Procedure for Generation and Purification of F(ab′)₂ Fragments:

The immobilized Pepsin was equilibrated by washing the resin with 0.5 mlof Digestion Buffer and centrifuging the column at 5000×g for 1 minute.The buffer was discarded afterwards. The desalting column was preparedby removing the storage solution and washing it with digestion buffer,centrifuging it each time afterwards at 1000×g for 2 minutes. 0.5 ml ofthe prepared IgG sample where added to the spin column tube containingthe equilibrated Immobilized Pepsin. Incubation time of the digestionreaction was done for 16 h on a tabletop rocker at 37° C. The column wascentrifuged at 5000×g for 1 minute to separate digest from theImmobilized Papain. Afterwards the resin was washed with 0.5 mL PBS andcentrifuged at 5000×g for 1 minute. The wash fraction was added to thedigested antibody that the total sample volume was 1.0 ml. The NAbProtein A Column was equilibrated with PBS and IgG Elution Buffer atroom temperature. The column was centrifuged for 1 minute to removestorage solution (contains 0.02% sodium azide) and equilibrated byadding 2 mL of PBS, centrifuge again for 1 minute and the flow-throughdiscarded. The sample was applied to the column and resuspended byinversion. Incubation was done at room temperature with end-over-endmixing for 10 minutes. The column was centrifuged for 1 minute, savingthe flow-through with the Fab fragments.

(References: Mariani, M., et al. (1991). A new enzymatic method toobtain high-yield F(ab′)2 suitable for clinical use from mouse IgG1.Mol. Immunol. 28: 69-77.; Beale, D. (1987). Molecular fragmentation:Some applications in immunology. Exp Comp Immunol 11:287-96.; Ellerson,J. R., et al. (1972). A fragment corresponding to the CH2 region ofimmunoglobulin G (IgG) with complement fixing activity. FEBS Letters24(3):318-22.; Kerbel, R. S. and Elliot, B. E. (1983). Detection of Fcreceptors. Meth Enzymol 93:113-147.; Kulkarni, P. N., et al. (1985).Conjugation of methotrexate to IgG antibodies and their F(ab′)2fragments and the effect of conjugated methotrexate on tumor growth invivo. Cancer Immunol Immunotherapy 19:211-4.; Lamoyi, E. (1986).Preparation of F(ab′)2 Fragments from mouse IgG of various subclasses.Meth Enzymol 121:652-663.; Parham, P., et al. (1982). Monoclonalantibodies: purification, fragmentation and application to structuraland functional studies of class I MHC antigens. J Immunol Meth53:133-73.; Raychaudhuri, G., et al. (1985). Human IgG1 and its Fcfragment bind with different affinities to the Fc receptors on the humanU937, HL-60 and ML-1 cell lines. Mol Immunol 22(9):1009-19.; Rousseaux,J., et al. (1980). The differential enzyme sensitivity of ratimmunoglobulin G subclasses to papain an pepsin. Mol Immunol 17:469-82.;Rousseaux, J., et al. (1983). Optimal condition for the preparation ofFab and F(ab′)2 fragments from monoclonal IgG of different rat IgGsubclasses. J Immunol Meth 64:141-6.; Wilson, K. M., et al. (1991).Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptideconjugate. J Immunol Meth 138:111-9.)

NT-H-Antibody Fragment Humanization

The antibody fragment was humanized by the CDR-grafting method (Jones,P. T., Dear, P. H., Foote, J., Neuberger, M. S., and Winter, G. (1986)Replacing the complementarity-determining regions in a human antibodywith those from a mouse. Nature 321, 522-525).

The Following Steps where Done to Achieve the Humanized Sequence:

Total RNA extraction: Total RNA was extracted from NT-H hybridomas usingthe Qiagen kit.

First-round RT-PCR: QIAGEN® OneStep RT-PCR Kit (Cat No. 210210) wasused. RT-PCR was performed with primer sets specific for the heavy andlight chains. For each RNA sample, 12 individual heavy chain and 11light chain RT-PCR reactions were set up using degenerate forward primermixtures covering the leader sequences of variable regions. Reverseprimers are located in the constant regions of heavy and light chains.No restriction sites were engineered into the primers.

Reaction Setup: 5×QIAGEN® OneStep RT-PCR Buffer 5.0 μl, dNTP Mix(containing 10 mM of each dNTP) 0.8 μl, Primer set 0.5 μl, QIAGEN®OneStep RT-PCR Enzyme Mix 0.8 μl, Template RNA 2.0 μl, RNase-free waterto 20.0 μl, Total volume 20.0 μl PCR condition: Reverse transcription:50° C., 30 min; Initial PCR activation: 95° C., 15 min

Cycling: 20 cycles of 94° C., 25 sec; 54° C., 30 sec; 72° C., 30 sec;Final extension: 72° C., 10 min

Second-round semi-nested PCR: The RT-PCR products from the first-roundreactions were further amplified in the second-round PCR. 12 individualheavy chain and 11 light chain RT-PCR reactions were set up usingsemi-nested primer sets specific for antibody variable regions.

Reaction Setup: 2×PCR mix 10 μl; Primer set 2 μl; First-round PCRproduct 8 μl; Total volume 20 μl; Hybridoma Antibody Cloning Report

PCR condition: Initial denaturing of 5 min at 95° C.; 25 cycles of 95°C. for 25 sec, 57° C. for 30 sec, 68° C. for 30 sec; Final extension is10 min 68° C.

After PCR is finished, run PCR reaction samples onto agarose gel tovisualize DNA fragments amplified. After sequencing more than 15 clonedDNA fragments amplified by nested RT-PCR, several mouse antibody heavyand light chains have been cloned and appear correct. Protein sequencealignment and CDR analysis identifies one heavy chain and one lightchain. After alignment with homologous human framework sequences theresulting humanized sequence for the variable heavy chain is thefollowing: see FIG. 6 (As the amino acids on positions 26, 40 and 55 inthe variable heavy chain and amino acid on position 40 in the variablelight are critical to the binding properties, they may be reverted tothe murine original. The resulting candidates are depicted below)(Padlan, E. A. (1991) A possible procedure for reducing theimmunogenicity of antibody variable domains while preserving theirligand-binding properties. Mol. Immunol. 28, 489-498.; Harris, L. andBajorath, J. (1995) Profiles for the analysis of immunoglobulinsequences: Comparison of V gene subgroups. Protein Sci. 4, 306-310.).

Annotation for the antibody fragment sequences (SEQ ID NO: 7-14): boldand underline are the CDR 1, 2, 3 in chronologically arranged; italicare constant regions; hinge regions are highlighted with bold lettersand the histidine tag with bold and italic letters; framework pointmutation have a grey letter-background.

Example 2 Effect of Selected Anti-ADM-Antibodies on Anti-ADM-Bioactivity

The effect of selected ADM-antibodies on ADM-bioactivity was tested inan human recombinant Adrenomedullin receptor cAMP functional assay(Adrenomedullin Bioassay).

Testing of Antibodies Targeting Human or Mouse Adrenomedullin in HumanRecombinant Adrenomedullin Receptor cAMP Functional Assay(Adrenomedullin Bioassay)

Materials:

-   Cell line: CHO-K1-   Receptor: Adrenomedullin (CRLR+RAMP3)-   Receptor Accession Number Cell line: CRLR: U17473; RAMP3: AJ001016

CHO-K1 cells expressing human recombinant adrenomedullin receptor(FAST-027C) grown prior to the test in media without antibiotic weredetached by gentle flushing with PBS-EDTA (5 mM EDTA), recovered bycentrifugation and resuspended in assay buffer (KRH: 5 mM KCl, 1.25 mMMgSO4, 124 mM NaCl, 25 mM HEPES, 13.3 mM Glucose, 1.25 mM KH2PO4, 1.45mM CaCl2, 0.5 g/1 BSA).

Dose response curves were performed in parallel with the referenceagonists (hADM or mADM).

Antagonist Test (96 well):

For antagonist testing, 6 μl of the reference agonist (human (5.63 nM)or mouse (0.67 nM) adrenomedullin) was mixed with 6 μl of the testsamples at different antagonist dilutions; or with 6 μl buffer. Afterincubation for 60 min at room temperature, 12 μl of cells (2,500cells/well) were added. The plates were incubated for 30 min at roomtemperature. After addition of the lysis buffer, percentage of DeltaFwill be estimated, according to the manufacturer specification, with theHTRF kit from Cis-Bio International (cat no 62AM2 PEB). hADM 22-52 wasused as reference antagonist.

Antibodies Testing cAMP-HTRF Assay

The anti-h-ADM antibodies (NT-H, MR-H, CT-H) were tested for antagonistactivity in human recombinant adrenomedullin receptor (FAST-027C) cAMPfunctional assay in the presence of 5.63 nM Human ADM 1-52, at thefollowing final antibody concentrations: 100 μg/ml, 20 μg/ml, 4 μg/ml,0.8 μg/ml, 0.16 μg/ml.

The anti-m-ADM antibodies (NT-M, MR-M, CT-M) were tested for antagonistactivity in human recombinant adrenomedullin receptor (FAST-027C) cAMPfunctional assay in the presence of 0.67 nM Mouse ADM 1-50, at thefollowing final antibody concentrations: 100 μg/ml, 20 μg/ml, 4 μg/ml,0.8 μg/ml, 0.16 μg/ml. Data were plotted relative inhibition vs.antagonist concentration (see FIGS. 3 a to 3 l). The maximal inhibitionby the individual antibody is given in table 3.

TABLE 3 Maximal inhibition of ADM Antibody bioactivity (ADM-Bioassay)(%) NT-H 38 MR-H 73 CT-H 100 NT-M FAB 26 NT-M FAB2 28 NT-M 45 MR-M 66CT-M 100 Non specific mouse IgG 0

Example 3 Data for Stabilization of hADM by the Anti-ADM Antibody

The stabilizing effect of human ADM by human ADM antibodies was testedusing a hADM immunoassay.

Immunoassay for the Quantification of Human Adrenomedullin

The technology used was a sandwich coated tube luminescence immunoassay,based on Acridinium ester labelling.

Labelled Compound (Tracer):

100 μg (100 ul) CT-H (1 mg/ml in PBS, pH 7.4, AdrenoMed AGGermany) wasmixed with 10 μl Acridinium NHS-ester (1 mg/ml in acetonitrile, InVentGmbH, Germany) (EP 0353971) and incubated for 20 min at roomtemperature. Labelled CT-H was purified by Gel-filtration HPLC onBio-Sil® SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified CT-Hwas diluted in (300 mmol/L potassiumphosphate, 100 mmol/L NaCl, 10mmol/L Na-EDTA, 5 g/L Bovine Serum Albumin, pH 7.0). The finalconcentration was approx. 800.000 relative light units (RLU) of labelledcompound (approx. 20 ng labeled antibody) per 200 μL. Acridiniumesterchemiluminescence was measured by using an AutoLumat LB 953 (BertholdTechnologies GmbH & Co. KG).

Solid Phase:

Polystyrene tubes (Greiner Bio-One International AG, Austria) werecoated (18 h at room temperature) with MR-H (AdrenoMed AG, Germany) (1.5μg MR-H/0.3 mL 100 mmol/L NaCl, 50 mmol/L TRIS/HCl, pH 7.8). Afterblocking with 5% bovine serum albumine, the tubes were washed with PBS,pH 7.4 and vacuum dried.

Calibration:

The assay was calibrated, using dilutions of hADM

(BACHEM AG, Switzerland) in 250 mmol/L NaCl, 2 g/L Triton X-100, 50 g/LBovine Serum Albumin, 20 tabs/L Protease Inhibitor Cocktail (RocheDiagnostics AG, Switzerland))

hADM Immunoassay:

-   50 μl of sample (or calibrator) was pipetted into coated tubes,    after adding labeleld CT-H (200 μl), the tubes were incubated for 4    h at 4° C. Unbound tracer was removed by washing 5 times (each 1 ml)    with washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100).

Tube-Bound Chemiluminescence was Measured by Using the LB 953

FIG. 4 shows a typical hADM dose/signal curve. And an hADM dose signalcurve in the presence of 100 μg/mL antibody NT-H.

NT-H did not affect the described hADM immunoassay.

Stability of Human Adrenomedullin:

Human ADM was diluted in human Citrate plasma (final concentration 10nM) and incubated at 24° C. At selected time points, the degradation ofhADM was stopped by freezing at −20° C. The incubation was performed inabsence and presence of NT-H (100 μg/ml). The remaining hADM wasquantified by using the hADM immunoassay described above.

FIG. 5 shows the stability of hADM in human plasma (citrate) in absenceand in the presence of NT-H antibody. The half life of hADM alone was7.8 h and in the presence of NT-H, the half life was 18.3 h. (2.3 timeshigher stability).

Example 4 Sepsis Mortality (Early Treatment) Animal Model

12-15 week old male C57Bl/6 mice (Charles River Laboratories, Germany)were used for the study. Peritonitis had been surgically induced underlight isofluran anesthesia. Incisions were made into the left upperquadrant of the peritoneal cavity (normal location of the cecum). Thececum was exposed and a tight ligature was placed around the cecum withsutures distal to the insertion of the small bowel. One puncture woundwas made with a 24-gauge needle into the cecum and small amounts ofcecal contents were expressed through the wound. The cecum was replacedinto the peritoneal cavity and the laparotomy site was closed. Finally,animals were returned to their cages with free access to food and water.500 μl saline were given s.c. as fluid replacement.

Application and Dosage of the Compound (NT-M, MR-M, CT-M)

Mice were treated immediately after CLP (early treatment). CLP is theabbreviation for cecal ligation and puncture (CLP).

Study Groups

Three compounds were tested versus: vehicle and versus control compoundtreatment. Each group contained 5 mice for blood drawing after 1 day forBUN (serum blood urea nitrogen test) determination. Ten further mice pereach group were followed over a period of 4 days.

Group Treatment (10 μl/g Bodyweight) Dose/Follow-Up:

-   1 NT-M, 0.2 mg/ml survival over 4 days-   2 MR-M, 0.2 mg/ml survival over 4 days-   3 CT-M, 0.2 mg/ml survival over 4 days-   4 non-specific mouse IgG, 0.2 mg/ml survival over 4 days-   5 control—PBS 10 μl/g bodyweight survival over 4 days

Clinical Chemistry

Blood urea nitrogen (BUN) concentrations for renal function weremeasured baseline and day 1 after CLP. Blood samples were obtained fromthe cavernous sinus with a capillary under light ether anaesthesia.Measurements were performed by using an AU 400 Olympus Multianalyser.The 4-day mortality is given in table 4. The average BUN concentrationsare given in table 5.

TABLE 4 4 day mortality survival (%) PBS 0 non-specific mouse IgG 0 CT-M10 MR-M 30 NT-M 70

TABLE 5 Average from 5 animals BUN pre CLP (mM) BUN day 1 (mM) PBS 8.023.2 non-specific mouse IgG 7.9 15.5 CT-M 7.8 13.5 MR-M 8.1 24.9 NT-M8.8 8.2

It can be seen from Table 4 that the NT-M antibody reduced mortalityconsiderably. After 4 days 70% of the mice survived when treated withNT-M antibody. When treated with MR-M antibody 30% of the animalssurvived and when treated with CT-M antibody 10% of the animals survivedafter 4 days. In contrast thereto all mice were dead after 4 days whentreated with unspecific mouse IgG. The same result was obtained in thecontrol group where PBS (phosphate buffered saline) was administered tomice.

The blood urea nitrogen or BUN test is used to evaluate kidney function,to help diagnose kidney disease, and to monitor patients with acute orchronic kidney dysfunction or failure.

The results of the S-BUN Test revealed that the NT-M antibody was themost effective to protect the kidney.

Sepsis Mortality (Late Treatment) Animal Model

12-15 week old male C57Bl/6 mice (Charles River Laboratories, Germany)were used for the study. Peritonitis had been surgically induced underlight isofluran anesthesia. Incisions were made into the left upperquadrant of the peritoneal cavity (normal location of the cecum). Thececum was exposed and a tight ligature was placed around the cecum withsutures distal to the insertion of the small bowel. One puncture woundwas made with a 24-gauge needle into the cecum and small amounts ofcecal contents were expressed through the wound. The cecum was replacedinto the peritoneal cavity and the laparotomy site was closed. Finally,animals were returned to their cages with free access to food and water.500 μl saline were given s.c. as fluid replacement.

Application and Dosage of the Compound (NT-M FAB2)

NT-M FAB2 was tested versus: vehicle and versus control compoundtreatment. Treatment was performed after full development of sepsis, 6hours after CLP (late treatment). Each group contained 4 mice and werefollowed over a period of 4 days.

Group Treatment (10 μl/g Bodyweight) Dose/Follow-Up:

Study groups

-   1 NT-M, FAB2 0.2 mg/ml survival over 4 days-   2 control: non-specific mouse IgG, 0.2 mg/ml survival over 4 days-   3 vehicle:—PBS 10 μl/g bodyweight survival over 4 days

TABLE 6 4 day mortality survival (%) PBS 0 Non-specific mouse IgG 0 NT-MFAB2 75

It can be seen from Table 6 that the NT-M FAB 2 antibody reducedmortality considerably. After 4 days 75% of the mice survived whentreated with NT-M FAB 2 antibody. In contrast thereto all mice were deadafter 4 days when treated with non-specific mouse IgG. The same resultwas obtained in the control group where PBS (phosphate buffered saline)was administered to mice.

Example 5 Incremental Effect of Anti-ADM Antibody in CLP-Animals on Topof Antibiotic Treatment and Circulation Stabilization Via Catecholaminesas Well as Regulation of Fluid Balance Animal Model

In this study male C57Bl/6 mice (8-12 weeks, 22-30 g) were utilized. Apolymicrobial sepsis induced by cecal ligation and puncture (CLP) wasused as the model for studying septic shock ((Albuszies G, et al: Effectof increased cardiac output on hepatic and intestinal microcirculatoryblood flow, oxygenation, and metabolism in hyperdynamic murine septicshock. Crit Care Med 2005; 33:2332-8), (Albuszies G, et al: The effectof iNOS deletion on hepatic gluconeogenesis in hyperdynamic murineseptic shock. Intensive Care Med 2007; 33:1094-101), (Barth E, et al:Role of iNOS in the reduced responsiveness of the myocardium tocatecholamines in a hyperdynamic, murine model of septic shock. CritCare Med 2006; 34:307-13), (Baumgart K, et al: Effect of SOD-1over-expression on myocardial function during resuscitated murine septicshock. Intensive Care Med 2009; 35:344-9),

(Baumgart K, et al: Cardiac and metabolic effects of hypothermia andinhaled H2S in anesthetized and ventilated mice. Crit Care Med 2010;38:588-95), (Simkova V, et al: The effect of SOD-1 over-expression onhepatic gluconeogenesis and whole-body glucose oxidation duringresuscitated, normotensive murine septic shock. Shock 2008; 30:578-84),(Wagner F, et al.: Inflammatory effects of hypothermia and inhaled H2Sduring resuscitated, hyperdynamic murine septic shock. Shock, im Druck),(Wagner F, et al: Effects of intravenous H2S after murine blunt chesttrauma: a prospective, randomized controlled trial. Crit Care 2011,submittes for publication)).

After weighing, mice were anesthetized by intraperitoneal injection of120 μg/g Ketamin, 1.25 μg/g Midazolam and 0.25 μg/g Fentanyl. During thesurgical procedure, body temperature was kept at 37-38° C. A 1 cmmidline abdominal section was performed to get access to the cecum. Thececum then was ligated with 3-0 silk tie close to the basis and a singlepuncture with a 18-gauge needle was applied. The cecum was returned andthe incision was closed again (4-0 tie). For the compensation ofperioperative loss of liquids, 0.5 ml lacted Ringer's solution with 1μg/g Buprenorphin as analgetic was injected subcutaneously in dorsaldermis. For antibiosis the mice received Ceftriaxon 30 μg/g andClindamycin 30 μg/g subcutaneously via the lower extremities.

After CLP surgery the animal were kept in an adequately heatedenvironment with water and food ad libitum.

The covering of liquid requirements were ensured by a dorsalsubcutaneous injections with 0.5 ml lactated ringer's solution with 4μg/g glucose and Buprenorphin 1 μg/g, which were applied in an 8 hourcycle, after short term anesthesia by isofluran. In addition, antibiosiswas maintained by subcutaneous injections of Ceftriaxon 30 μg/g andClindamycin 30 μg/g via the lower extremities.

Dosing of Test Substances Early Treatment

Immediately after the CLP surgery and closing of the incision, the testsubstance antibody NT-M was applied in a concentration of 500 μg/ml inphosphate buffered saline (PBS) via injection into the penis vein for adose of 2 mg per kg body weight (dose volume 88-120 μl) (5 animals).

Late Treatment

After full Sepsis development, 15.5 h after CLP surgery, animals wereanesthetized as described above and NT-M was applied in a concentrationof 500 μg/ml in phosphate buffered saline (PBS) via injection into thepenis vein for a dose of 2 mg per kg body weight (dose volume 88-120 μl)(3 animals).

The control group (6 animals) received a corresponding amount of thevehicle PBS solution without antibody (4 μl/g, 88-120 μl) immediatelyafter CLP surgery.

Study Groups and Experimental Setting

Murine septic shock model under intensive care monitoring:

Three groups with 3, 5 and 6 animals were monitored. Group 1 (5 animals)received the antibody NT-M 15.5 h after CLP, group 2 received theantibody NT-M immediately after CLP surgery and group 3 received acomparable amount of PBS (4 μl/g). 16 hour incubation post CLP (to allowthe polymicrobial sepsis to progress), the experiment was continued withmonitoring and interventions comparable to an intensive medical careregime. Therefore, after weighing the animals were anesthetized asdescribed in the CLP surgery part (except the late treated animals,which were anesthized before treatment). Body temperature was maintainedat 37-38° C. for the rest of the experiment. After a tracheotomy andintubation, respiration was monitored and supported by laboratory animallung ventilator Flexivent®, (Emka Technologies, FiO2 0.5, PEEP 10 H2O,VT 8 μl/g, I:E 1:1.5, AF 70-140 depending on temperature).

Anesthesia was maintained throughout the experiment via the cannulatedvena jugularis externa dextra with a continuous infusion of Ketamin 30μg/g×h and Fentanyl 0.3 μg/g×h. Furthermore, the right aorta carotiscommunis was cannulated for continuous monitoring of heart rate and themean arterial pressure (MAP). The mean arterial pressure was maintainedat MAP >65 mmHg via intravenous (V. jugularis) infusion of colloids (80μL/g×h, Hextend®) and, if needed, Noradrenalin dissolved in colloids asvasopressor. Blood samples (120 μl) were taken via the cannulated A.carotis at 0 and 4 hours for determination of creatinine. The bladderwas punctured and urine was collected via a bladder catheter. Theexperiment was either terminated after 6 hours or prior to this, if theMAP >65 mmHg (V. jugularis) could not be maintained with thevasorpressor dosing.

Measured Parameters

The following parameters were measured and analyzed: Total consumptionof noradrenalin (μg NA/g), consumption rate of noradrenalin (μg NA/g/h),total volume of urine collected during the experiment, creatinineconcentration (μg/mL) at the end of the experiment and mean creatinineclearance (μL/min).

TABLE 7 Total consumption consumption rate of Noradrenalin ofNoradrenalin (μg NA/g) (μg NA/g/h) (Average) (Average) Control (mouseIgG) (N = 6) 0.17 μg/g 0.032 μg/h/g NT-M (N = 5) early treatment 0.07μg/g 0.012 μg/h/g Relative change (early treatment,   59% 62.5%amelioration)   (59%) (62.5%) NT-M (N = 3) late treatment 0.04 μg/g0.0075 μg/h/g  Relative change (late treatment, 76.5% 76.5%amelioration) (76.5%) (76.5%)

The catecholamine requirement was measured after administration ofeither non specific mouse IgG to a total of 6 mice as control group,NT-murine antibody to a group of 5 mice immediately after CLP (earlytreatment) or NT-murine antibody to a group of 3 mice 15.5 h after CLP(late treatment).

The reduction of the catecholamine requirement is a measure for thestabilization of the circulation. Thus, the data show that the ADMantibody, especially the NT-M antibody, leads to a considerablestabilization of the circulation and to a considerable reduction of thecatecholamine requirement. The circulation-stabilizing effect was givenin early treatment (immediately after CLP) and treatment after fullsepsis development (late treatment) (see FIG. 7).

Regulation of Fluid Balance

More positive fluid balance both early in resuscitation and cumulativelyover 4 days is associated with an increased risk of mortality in septicshock. The control of the liquid balance is of utmost importance for thecourse of disease of patients having sepsis. (s. Boyd et al, 2011).Controlling the liquid balance of critical ill patients remains as asubstantial challenge in intensive care medicine. As can be seen intable 8 treatment of mice after CLP (experimental procedures see “AnimalModel”) with NT-M antibody lead to an enhancement of the total volume ofurine excreted. The urine secreted was approx. three times higher inNT-M-treated animals compared to non-treated mice. The positivetreatment effect was given in early- and in late treatment. The fluidbalance was improved by about 20-30%, also in both, early and latetreatment. Thus, the data show that the use of ADM antibody, especiallythe use of NT ADM antibody, is favorable for regulating the fluidbalance in patients. (see table 8 and FIGS. 8 and 9).

TABLE 8 Urine average Fluid balance volume/g body average volume/gweight body weight Control (mouse IgG) 0.042 ml/g 0.23 ml/g (N = 6) NT-Mearly (N = 5) 0.12 ml 0.18 ml/g Relative change early +186%    −21.7%%treatment NT-M late (N = 3) 0.125 ml 0.16 ml/g Relative change late+198% −30.5% treatment

Improvement of Kidney Function

The combination of acute renal failure and sepsis is associated with a70 percent mortality, as compared with a 45 percent mortality amongpatients with acute renal failure alone. (Schrier and Wang, “Mechanismsof Disease Acute Renal Failure and Sepsis”; The New England Journal ofMedicine; 351:159-69; 2004). Creatinine concentration and creatinineclearance are standard laboratory parameters for monitoring kidney(dys)function (Jacob, “Acute Renal Failure”, Indian J. Anaesth.; 47 (5):367-372; 2003). Creatinine and creatinine clearance data from abovedescribed animal experiment (early treatment) are given in Table 9.

TABLE 9 Kidney function: creatinine mean creatinine concentrationclearance (μg/mL) (μL/min) control mouse IgG (MW) 2.6 μg/ml 174 μl/minNT-M (MW) 1.5 μg/ml 373 μl/min Relative change −42% +114% (amelioration) (42%)  (114%)

In comparision to control septic animals, the creatinine concentrationwas lowered by 42% and the creatinine clearance was improved by morethan 100% as a result of NT-M treatment (Table 9). The data show thatthe administration of ADM-antibody, especially NT-M, leads to animprovement of kidney function.

Improvement of Liver Inflammatory Status

Liver tissue for control and early treated animals was homogenized andlysed in lysing buffer. For cell extract preparation, cells wereresuspended, lysed on ice, and centrifuged. The supernatant (proteinextract) was stored at −80° C. Activation of nuclear factorkappa-light-chain gene enhancer in B cells (NF-κB) was determined aspreviously described using an electrophoretic mobility shift assay(EMSA) 1.2. Cell extracts (10 μg) were incubated on ice withpoly-doxy-inosinic-deoxy-cytidylic acid (poly-dI-dC) and 32P-labeleddouble stranded oligonucleotide (Biomers, Ulm, Germany) containing theNF-κB (HIV κBsite) (5′-GGATCCTCAACAGAGGGGACTTTCCGAGGCCA-3′). Complexeswere separated in native polyacrylamide gels, dried and exposed to X-rayfilms. A phosphorimager and image analyzer software (AIDA ImageAnalyzer; Raytest) was used to quantify the radioactively labeled NF-κBby densitometry. For comparison between individual gels, the intensityof each band was related to that of simultaneously loaded controlanimals which had not undergone surgical instrumentation and CLP.Therefore, the EMSA data are expressed as fold increase over controlvalues. Statistics: All data are presented as median (range) unlessotherwise stated differences between the two groups were analyzed withthe Mann-Whitney rank sum test for unpaired samples. Results: Theanimals treated with NT-M presented with significantly attenuated livertissue NF-κB activation (2.27 (1.97-2.53)) compared to vehicle animals(2.92 (2.50-3.81)) (p<0.001) (see FIG. 10).

REFERENCES

-   1. Wagner F, Wagner K, Weber S, Stahl B, Knöferl M W, Huber-Lang M,    Seitz D H, Asfar P, Calzia E, Senftleben U, Gebhard F, Georgieff M,    Radermacher P, Hysa V: Inflammatory effects of hypothermia and    inhaled H2S during resuscitated, hyperdynamic murine septic shock.    Shock 2011; 35(4):396-402-   2. Wagner F, Scheuerle A, Weber S, Stahl B, McCook O, Knöferl M W,    Huber-Lang M, Seitz D H, Thomas J, Asfar P, Szabó C, Möller P,    Gebhard F, Georgieff M, Calzia E, Radermacher P, Wagner K:    Cardiopulmonary, histologic, and inflammatory effects of intravenous    Na2S after blunt chest trauma-induced lung contusion in mice. J    Trauma 2011; 71(6):1659-67.

Example 6 In Vivo Side Effect Determination of Antibody NT-M

12-15 week old male C57Bl/6 mice (Charles River Laboratories, Germany)were used for the study. 6 mice were treated with (10 ul/g bodyweight)dose of NT-M, 0.2 mg/ml. As control, 6 mice were treated with (10 μl/gbody weight) PBS. Survival and physical condition was monitored for 14days. The mortality was 0 in both groups, there were no differences inphysical condition between NT-M and control group.

Example 7 Gentamicin-Induced Nephrotoxicity

A non-septic acute kidney injury model has been established, which makesuse of the nephrotoxicity induced by Gentamicin (Chiu P J S. Models usedto assess renal functions. Drug Develop Res 32:247-255, 1994.). Thismodel was used to assess whether treatment with anti-Adrenomedullinantibody can improve kidney function.

The experiment was performed as follows:

Effect of a NT-M on Gentamicin-Induced Nephrotoxicity in Rats StudyDesign: Test Conc Dosage Rats^(d) Group Article Route mg/ml ml/kg mg/kg(Male) 1 Gentamicin^(a) + IV NA × 4^(c) 8 vehicle^(b) 2 Gentamicin^(a) +IV ×4^(c) 8 NT-M ^(a)Gentamicin at 120 mg/kg intramuscularly for 7 days(days 0-6). ^(b)Vehicle; injected intravenously (i.v.) 5 min beforegentamicin on Day 0, followed by injections on Days 2, 4, and 6.^(c)NT-M at 4 mg/kg was injected intravenously (i.v.) 5 min beforegentamicin on Day 0, followed by 2 mg/kg i.v. on Days 2, 4, and 6.^(d)Plasma samples were collected in EDTA tubes (Days 1 and 3 beforeTest and Control article: 100 μl; Day 7: 120 μl. 24 h urine collectionon ice is initiated after gentamicin on Day 0, followed by Days 2 and 6;blood collection on days 1, 3, and 7.

Groups of 8 male Sprague-Dawley rats weighing 250±20 g were employed.Animals were challenged with gentamicin at 120 mg/kg i.m. for sevenconsecutive days (Groups 1 and 2). Test compound (anti-adrenomedullinantibody NT-M) and vehicle (phosphate buffered saline) were injectedintravenously 5 min before gentamicin on day 0, followed by injection ondays 2, 4, and 6. Body weights and clinical signs were monitored daily.Twenty-four (24) hour urine collections on ice were performed on Days 0,2, and 6. Urine specimens were assayed for concentrations of Na+ and K+,and creatinine. Blood samples for clinical chemistry were collected onDays 1 (before gentamicin), 3 (before gentamicin), and 7. Serumelectrolytes (Na+ and K+), creatinine, and BUN were the primary analytesthat were monitored for assessing renal function. Plasma samples werecollected in EDTA tubes (Days 1 and 3:100 μl; Day 7:120 μl). Creatinineclearance was calculated. Urine volume, urinary electrolytes, andcreatinine are expressed as amount excreted per 100 g of animal bodyweight. All animals were sacrificed on Day 7. Kidneys were weighed.

Urine collection. The animals were placed in individual cages whereurine was collected for 24 h on Day 0, Day 2, and Day 6. Urine volume,urinary Na+, K+, and creatinine were measured.

Endogenous creatinine clearance was calculated as follows:

CCr(ml/24 h)=[UCr(mg/ml)×V(ml/24 h)]/SCr(mg/ml)

24-hr urinary excretion of sodium (Na+) was calculated as follows:

UNaV(μEq/24 h)=UNa(μEq/ml)×V(ml/24 h)

24-hr urinary excretion of NAG and NGALwas similarly calculated.

The fractional excretion of Na⁺ (FE_(Na)), or percentage of the filteredsodium that is excreted into the final urine, is a measure of tubularNa⁺ reabsorptive function. It was computed as follows:

FE_(Na)(%)=100×[U_(Na)(μEq/ml)×V(ml/24 h)]/P_(Na)(μEq/ml)×C_(Cr)(ml/24h)

Treatment with anti-Adrenomedullin antibody improved several measures ofkidney function on day 7 as compared to vehicle: serum creatinine 1.01mg/dL (NT-M) vs 1.55 mg/dL (vehicle) (FIG. 11), BUN 32.08 mg/dL(NT-M)vs. 52.41 mg/dL (vehicle) (FIG. 12), endogenous creatinine clearance934.43 mL/24 h (NT-M) vs. 613.34 mL/24 h (vehicle) (FIG. 13), fractionalsecretion of Na⁺0.98% (NT-M) vs. 1.75% (vehicle) (FIG. 14).

Example 8

In the mice CLP model described above, the effect of treatment withanti-adrenomedullin antibody NT-M on several parameters of kidneyfunction was investigated.

NT-M caused a three- and two-fold higher diuresis and creatinineclearance, respectively, ultimately resulting in lower creatinine, urea,and NGAL blood concentrations at the end of the experiment (see Table10). Moreover, keratinocyte-derived chemokine (KC) concentrations in thekidney were significantly lowered by treatment with NT-M (FIG. 15).

TABLE 10 Parameters of kidney function in the vehicle-(n = 11) andNT-M-treated (n = 9) animals. Blood concentrations were measured insamples taken at the end of the experiment. NGAL = neutrophilgelatinase- associated lipocalin. All data are median (quartiles).Vehicle NT-M p-Value Urine output  4.4 (3.5; 16.5)  15.2 (13.9; 22.5)0.033 [μL · g⁻¹ · h⁻¹] Creatinine clearance 197 (110; 301) 400 (316;509) 0.006 [μL · min⁻¹] Creatinine [μg · mL⁻¹]  1.83 (1.52; 3.04)  1.28(1.20; 1.52) 0.010 Urea [μg · mL⁻¹] 378 (268; 513) 175 (101; 184) 0.004NGAL [μg · mL⁻¹] 16 (15; 20)  11 (10; 13)  0.008

The experiments were performed as follows:

Creatinine, Urea, and Neutrophil Gelatinase-Associated Lipocalin (NGAL)

Blood NGAL concentrations were measured using a commercial ELISA (mouseNGAL, RUO 042, BioPorto Diagnostics A/S, Denmark, Gentofte). Urea andcreatinine concentrations were measured with a capillary column(Optima-5MS, Macherey-Nagel, Diiren, Germany) gas chromatography/massspectrometry system (Agilent 5890/5970, Boblingen, Germany) using²H₃-creatinine (CDN isotopes, Pointe-Claire, QU, Canada) and methyl-urea(FlukaChemikalien, Buchs, Switzerland) as internal standards. Afterdeproteinization with acetonitrile, centrifugation and evaporation todryness, the supernatant was reconstituted in formic acid, and extractedover a weak anion exchange column (WCX, Phenomenex, Aschaffenburg,Germany). Acetonitrile plus N,O-Bis(trimethylsilyl)trifluoroacetamideand N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide allowedformation of the urea tert-butyl-dimethylsilyl- and thecreatininetrimethylsilyl-derivatives, respectively. Ions m/z 231 and245, and m/z 329 and 332 were monitored for urea and creatinine analytesand internal standards, respectively. From the urine output and theplasma and urine creatinine concentrations creatinine clearance wascalculated using the standard formula.

Sample Preparation

The kidney which was stored at −80° C. was disrupted with a homogenizerin PBS and lysed with a 2-fold concentrated buffer for a whole celllysate (100 mM Tris pH 7.6; 500 mM NaCl; 6 mM EDTA; 6 mM EGTA; 1%Triton-X-100; 0.5% NP 40; 10% Glycerol; Protease-Inhibitors(β-Glycerolphosphate 2 mM; DTT 4 mM; Leupeptine 20 μM;Natriumorthovanadate 0.2 mM)) and subsequently centrifuged. The wholecell lysate was obtained out of the supernatant; the pellet consistingof cell remnants was discarded. The amount of protein was determinedphotometrically with a commercially available protein assay (Bio-Rad,Hercules, Calif.) and the specimens were adjusted in the way that thefinal protein concentration was 4 μg/μl. The samples for the Multiplex-and EMSA analysis were diluted 1:1 with EMSA buffer (10 mM Hepes; 50 mMKCl; 10% Glycerol; 0.1 mM EDTA; 1 mM DTT), the samples for the immunoblots 1:1 with 2-fold Sample Buffer (2% SDS; 125 mM Tris-HCL (pH 6.8 at25° C.); 10% Glycerol; 50 mM DTT; 0.01% Bromophenol blue).

Levels of keratinocyte-derived chemokine (KC) concentrations weredetermined using a mouse multiplex cytokine kit (Bio-Plex Pro CytokineAssay, Bio-Rad, Hercules, Calif.), the assay was performed by using theBio-plex suspension array system with the manufacturer's instructions(see also Wagner F, Wagner K, Weber S, Stahl B, Knöferl M W, Huber-LangM, Seitz D H, Asfar P, Calzia E, Senftleben U, Gebhard F, Georgieff M,Radermacher P, Hysa V. Inflammatory effects of hypothermia and inhaledH2S during resuscitated, hyperdynamic murine septic shock. Shock 2011;35:396-402; and Wagner F, Scheuerle A, Weber S, Stahl B, McCook O,Knöferl M W, Huber-Lang M, Seitz D H, Thomas J, Asfar P, Szabó C, MöllerP, Gebhard F, Georgieff M, Calzia E, Radermacher P, Wagner K.Cardiopulmonary, histologic, and inflammatory effects of intravenousNa2S after blunt chest trauma-induced lung contusion in mice. J Trauma2011; 71:1659-1667). In brief, the appropriate cytokine standards andsamples were added to a filter plate. The samples were incubated withantibodies chemically attached to fluorescent-labeled micro beads.Thereafter, premixed detection antibodies were added to each well, andsubsequently, streptavidin-phycoerythrin was added. Beads were thenre-suspended, and the cytokines reaction mixture was quantified usingthe Bio-Plex protein array reader. Data were automatically processed andanalyzed by Bio-Plex Manager Software 4.1 using the standard curveproduced from recombinant cytokine standards. Levels below the detectionlimit of the assays were set to zero for statistical purposes.

Example 9

In the mice CLP model described above, the effect of treatment withanti-adrenomedullin antibody NT-M on the liver was investigated.

NT-M caused a significant lowering of keratinocyte-derived chemokine(KC) concentrations in the liver (FIG. 16).

Measurement of keratinocyte-derived chemokine (KC) was done analogous toexample 8 (kidney).

Example 10

In the mice CLP model described above, the effect of treatment withanti-adrenomedullin antibody NT-M on several cytokines and chemokinesinthe blood circulation (plasma) was investigated.

Cytokine and Chemokine Concentrations

Plasma levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6,monocyte chemoattractant protein (MCP)-1, and keratinocyte-derivedchemokine (KC) concentrations were determined using a mouse multiplexcytokine kit (Bio-Plex Pro Cytokine Assay, Bio-Rad, Hercules, Calif.),the assay was performed by using the Bio-plex suspension array systemwith the manufacturer's instructions (see also Wagner F, Wagner K, WeberS, Stahl B, Knöferl M W, Huber-Lang M, Seitz D H, Asfar P, Calzia E,Senftleben U, Gebhard F, Georgieff M, Radermacher P, Hysa V.Inflammatory effects of hypothermia and inhaled H2S during resuscitated,hyperdynamic murine septic shock. Shock 2011; 35:396-402; and Wagner F,Scheuerle A, Weber S, Stahl B, McCook O, Knöferl M W, Huber-Lang M,Seitz D H, Thomas J, Asfar P, Szabó C, Möller P, Gebhard F, Georgieff M,Calzia E, Radermacher P, Wagner K. Cardiopulmonary, histologic, andinflammatory effects of intravenous Na2S after blunt chesttrauma-induced lung contusion in mice. J Trauma 2011; 71:1659-1667).

In brief, the appropriate cytokine standards and samples were added to afilter plate. The samples were incubated with antibodies chemicallyattached to fluorescent-labeled micro beads. Thereafter, premixeddetection antibodies were added to each well, and subsequently,streptavidin-phycoerythrin was added. Beads were then re-suspended, andthe cytokines reaction mixture was quantified using the Bio-Plex proteinarray reader. Data were automatically processed and analyzed by Bio-PlexManager Software 4.1 using the standard curve produced from recombinantcytokine standards. Levels below the detection limit of the assays wereset to zero for statistical purposes.

Plasma levels and kidney tissue concentrations of tumor necrosis factor(TNF)-α, interleukin (IL)-6 and IL-10, monocyte chemoattractant protein(MCP)-1, and keratinocyte-dervived chemokine (KC) were determined usinga commercially available “Multiplex Cytokine Kit” (Bio-Plex ProPrecision Pro Cytokine Assay, Bio-Rad, Hercules, Calif.), which allowsto collect several parameters out of one single sample. The individualwork steps of the assay were performed according to the manufacturer'sinstructions (see also Wagner F, Wagner K, Weber S, Stahl B, Knöferl MW, Huber-Lang M, Seitz D H, Asfar P, Calzia E, Senftleben U, Gebhard F,Georgieff M, Radermacher P, Hysa V. Inflammatory effects of hypothermiaand inhaled H2S during resuscitated, hyperdynamic murine septic shock.Shock 2011; 35:396-402; and Wagner F, Scheuerle A, Weber S, Stahl B,McCook O, Knöferl M W, Huber-Lang M, Seitz D H, Thomas J, Asfar P, SzabóC, Möller P, Gebhard F, Georgieff M, Calzia E, Radermacher P, Wagner K.Cardiopulmonary, histologic, and inflammatory effects of intravenousNa2S after blunt chest trauma-induced lung contusion in mice. J Trauma2011; 71:1659-1667).

In brief, the fluorescence-labed microspheres (“beads”) were added to a96-well plate, followed by two washing steps, the addition of internalstandards and the addition of plasma- and kidney homogenate samples.During the subsequent incubation the single cytokines bind to theantibodies attached to polystyrene-beads. After the addition of thecytokine-specific biotin-labeled antibodies, which are for the detectionof the single cytokines, and an additional incubation time, subsequentlyphycoerythrin-labeled streptavidine was added. After an additionalincubation time, beads were then resuspended, and the plates could bemeasured with a specific flow cytometer (Bio-Plex suspension arraysystem, Bio-Rad, Hercules, Calif.). Data were automatically processedand analyzed by Bio-Plex Manager Software 4.1 using the standard curveproduced from recombinant cytokine standards. For the plasma levels theconcentration was provided in pg*mL⁻¹, the concentration of the kidneyhomogenates were converted to the appropriate protein concentration andprovided in pg*mg⁻¹ protein.

NT-M caused a significant lowering of plasma concentrations of IL-6(FIG. 17), IL-10 (FIG. 18), keratinocyte-derived chemokine (KC) (FIG.19), monocyte chemoattractant protein-1 (MCP-1) (FIG. 20), TNF-alpha(FIG. 21).

Example 11 Ischemia/Reperfusion-Induced Acute Kidney Injury

Another non-septic acute kidney injury model has been established, whereacute kidney injury is induced by ischemia/reperfusion (Nakamoto M,Shapiro J I, Shanley P F, Chan L, and Schrier R W. In vitro and in vivoprotective effect of atriopeptin III on ischemic acute renal failure. JClinlnvest 80:698-705, 1987., Chintala M S, Bernardino V, and Chiu P JS. Cyclic G M P but not cyclic AMP prevents renal platelet accumulationfollowing ischemia-reperfusion in anesthetized rats. J PharmacolExpTher271:1203-1208, 1994). This model was used to assess whether treatmentwith anti-adrenomedullin antibody can improve kidney function.

The experiment was performed as follows:

Effect of a NT-M on Acute Kidney Injury Induced by Ischemia/Reperfusionin Rats Study Design: Test Conc Dosage Rats Group Article Route mg/mlml/kg mg/kg (Male) 1 I-R + vehicle^(a) IV 5 NA × 3 8 2 I-R + NT-M IV 5×3^(b) 8 ^(a)vehicle; injected intravenously (i.v.) 5 min beforereperfusion on day 0, followed by injections on days 1 and 2. ^(b)NT-Mat 4 mg/kg was injected intravenously (i.v.) 5 min before reperfusion onday 0, followed by 2 mg/kg i.v. each on days 1 and 2. ^(c)Urinecollection on days −1, 0, 1 and 2, with blood chemistry and urineanalysis on days 0, 1, 2 and 3, respectively. Plasma samples werecollected in EDTA tubes (Days 0 (immediate before surgery), 1, 2: 100μl, before vehicle or TA; Day 3: 120 μl. Clinical observations: dailybefore surgery, following surgery and throughout treatment.

Groups of 8 male Sprague-Dawley rats weighing 250 to 280 g were used.The animals were kept on a 12-hr light/dark cycle and receive a standarddiet with distilled water ad libitum. The animals receive fluidsupplements (0.9% NaCl and 5% dextrose/1:1, 10 ml/kg p.o.) 30 min priorto surgery (day 0). The rats were anaesthetized with pentobarbital (50mg/kg, i.p.). The abdominal cavity was exposed via a midline incision,followed by intravenous administration of heparin (100 U/kg, i.v.) andboth renal arteries were occluded for 45 min by using vascular clamps.Immediately after removal of the renal clips, the kidneys were observedfor additional 1 min to ensure color change indicating bloodreperfusion. The test compound (NT-M) and vehicle (phosphate bufferedsaline) were injected intravenously 5 min before reperfusion, followedby daily injection on days 1 and 2.

Urine collection. The 24-h urine collection on ice was initiated at 24 hbefore ischemia/reperfusion on day −1 (−24 h to 0 h), and day 0 (0-24h), day 1 (24-48 h) and day 2 (48-72 h) after reperfusion,

Blood collection: 0.4 ml blood was collected through the tail vein intoEDTA tubes at 0 h (before I RI surgery), 24 h (before vehicle or TA), 48h (before vehicle or TA) and 72 h for determination of plasmacreatinine/Na+/K+, and BUN; 2 ml blood was collected through venal cavaterminally.

The animals were placed in individual cages where urine was collectedfor 24 h day −1 (−24 h-0 h), day 0 (0-24 h), day 1 (24-48 h) and day 2(48-72 h) after reperfusion on day 0. Urine volume, urinary Na+, K+, andcreatinine were measured.

The creatinine clearance (CCr) was calculated as follows:

CCr(ml/24 h)=[UCr(mg/ml)×V(ml/24 h)]/PCr(mg/ml)

The 24-hr urinary excretion of sodium (Na+) was calculated as follows:

UNaV(μEq/24 h)=UNa(μEq/ml)×V(ml/24 h)

The fractional excretion of Na+(FENa), or percentage of the filteredsodium that is excreted into the final urine, is a measure of tubularNa+ reabsorptive function. It was computed as follows:

FENa(%)=100×[UNa(μEq/ml)×V(ml/24 h)]/PNa(μEq/ml)×CCr(ml/24 h)

Treatment with anti-Adrenomedullin antibody improved several measures ofkidney function:

Blood urea nitrogen (BUN) showed a strong increase in the vehicle group(0 h: 17.49 mg/dL, 24 h: 98.85 mg/dL, 48 h: 109.84 mg/dL, 72 h: 91.88mg/dL), which was less pronounced with NT-M treatment (0 h: 16.33 mg/dL,24 h: 84.2 mg/dL, 48 h: 82.61 mg/dL, 72 h: 64.54 mg/dL) (FIG. 22).

Serum creatinine developed similarily: Vehicle group (0 h: 0.61 mg/dL,24 h: 3.3 mg/dL, 48 h: 3.16 mg/dL, 72 h: 2.31 mg/dL), NT-M group: (0 h:0.59 mg/dL, 24 h: 2.96 mg/dL, 48 h: 2.31 mg/dL, 72 h: 1.8 mg/dL) (FIG.23).

The endogenous creatinine clearance dropped massively on day one andthereafter improved better in the NT-M group than in the vehicle group.Vehicle group: (0 h: 65.17 mL/h, 24 h: 3.5 mL/h, 48 h: 12.61 mL/h, 72 h:20.88 mL/h), NT-M group: (0 h: 70.11 mL/h, 24 h: 5.84 mL/h, 48 h: 21.23mL/h, 72 h: 26.61 mL/h) (FIG. 24).

FIGURE DESCRIPTION

FIG. 1 a:

Illustration of antibody formats—Fv and scFv-Variants

FIG. 1 b:

Illustration of antibody formats—heterologous fusions and bifunctionalantibodies

FIG. 1 c:

Illustration of antibody formats—bivalental antibodies and bispecificantibodies

FIG. 2:

-   hADM 1-52 (SEQ ID No. 21)-   mADM 1-50 (SEQ ID No. 22)-   aa 1-21 of human ADM (SEQ ID No. 23)-   aa 1-42 of human ADM (SEQ ID No. 24)-   aa 43-52 of human ADM (SEQ ID No. 25)-   aa 1-14 of human ADM (SEQ ID NO: 26)-   aa 1-10 of human ADM (SEQ ID NO: 27)-   aa 1-6 of human ADM (SEQ ID NO: 28)-   aa 1-32 of human mature human ADM (SEQ ID NO: 29)-   aa 1-40 of mature murine ADM (SEQ ID NO: 30)-   aa 1-31 of mature murine ADM (SEQ ID NO: 31)

FIG. 3:

-   a: Dose response curve of human ADM. Maximal cAMP stimulation was    adjusted to 100% activation-   b: Dose/inhibition curve of human ADM 22-52 (ADM-receptor    antagonist) in the presence of 5.63 nM hADM.-   c: Dose/inhibition curve of CT-H in the presence of 5.63 nM hADM.-   d: Dose/inhibition curve of MR-H in the presence of 5.63 nM hADM.-   e: Dose/inhibition curve of NT-H in the presence of 5.63 nM hADM.-   f: Dose response curve of mouse ADM. Maximal cAMP stimulation was    adjusted to 100% activation-   g: Dose/inhibition curve of human ADM 22-52 (ADM-receptor    antagonist) in the presence of 0.67 nM mADM.-   h: Dose/inhibition curve of CT-M in the presence of 0.67 nM mADM.-   i: Dose/inhibition curve of MR-M in the presence of 0.67 nM mADM.-   j: Dose/inhibition curve of NT-M in the presence of 0.67 nM mADM.-   k: shows the inhibition of ADM by F(ab)2 NT-M and by Fab NT-M-   l: shows the inhibition of ADM by F(ab)2 NT-M and by Fab NT-M

FIG. 4:

This figure shows a typical hADM dose/signal curve. And an hADM dosesignal curve in the presence of 100 μg/mL antibody NT-H.

FIG. 5:

This figure shows the stability of hADM in human plasma (citrate) inabsence and in the presence of NT-H antibody.

FIG. 6:

Alignment of the Fab with homologous human framework sequences

FIG. 7:

This figure shows the Noradrenalin requirements for early and latetreatment with NT-M

FIG. 8:

This figure shows urine production after early and late treatment withNT-M

FIG. 9:

This figure shows the fluid balance after early and late treatment withNT-M

FIG. 10:

Liver tissue activation of nuclear factor kappa-light-chain geneenhancer in B cells (NF-κB) analyzed by electophoretic mobility shiftassay (EMSA). # depicts p<0.001 vs. vehicle.

FIG. 11:

Development of serum creatinine over time. Mean+/−SEM are shown.

FIG. 12:

Development of blood urea nitrogen (BUN) over time. Mean+/−SEM areshown.

FIG. 13:

Development of endogenous creatinine clearance over time. Mean+/−SEM areshown.

FIG. 14:

Development of fractional secretion of Na⁺ over time. Mean+/−SEM areshown.

FIG. 15:

Keratinocyte-derived chemokine (KC) levels determined in relation to thetotal kidney protein extracted. The white box-plot shows resultsobtained with vehicle, the grey box-plot shows results obtained aftertreatment with NT-M.

FIG. 16:

Keratinocyte-derived chemokine (KC) levels determined in relation to thetotal liver protein extracted. The white box-plot shows results obtainedwith vehicle, the grey box-plot shows results obtained after treatmentwith NT-M.

FIG. 17:

Plasma IL-6 levels. The white box-plot shows results obtained withvehicle, the grey box-plot shows results obtained after treatment withNT-M.

FIG. 18:

Plasma IL-10 levels. The white box-plot shows results obtained withvehicle, the grey box-plot shows results obtained after treatment withNT-M.

FIG. 19:

Plasma keratinocyte-derived chemokine (KC) levels. The white box-plotshows results obtained with vehicle, the grey box-plot shows resultsobtained after treatment with NT-M.

FIG. 20:

Plasma monocyte chemoattractant protein-1 (MCP-1) levels. The whitebox-plot shows results obtained with vehicle, the grey box-plot showsresults obtained after treatment with NT-M.

FIG. 21:

Plasma TNF-alpha levels. The white box-plot shows results obtained withvehicle, the grey box-plot shows results obtained after treatment withNT-M.

FIG. 22:

Development of blood urea nitrogen (BUN) over time. Mean+/−SEM areshown.

FIG. 23:

Development of serum creatinine over time. Mean+/−SEM are shown.

FIG. 24:

Development of endogenous creatinine clearance over time. Mean+/−SEM areshown.

1. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient for prevention or reduction of organ dysfunction or prevention of organ failure in said patient.
 2. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said anti-ADM antibody or said anti-adrenomedullin antibody fragment or said anti-ADM non-Ig scaffold is a non-neutralizing ADM antibody or a non-neutralizing adrenomedullin antibody fragment or a non-neutralizing ADM non-Ig scaffold.
 3. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said organ is kidney or liver.
 4. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said organ is heart.
 5. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said antibody or fragment or scaffold binds to the N-terminal part (aa 1-21) of adrenomedullin.
 6. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said antibody or antibody fragment or non-Ig scaffold is monospecific.
 7. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said antibody or fragment or scaffold exhibits a binding affinity to ADM of at least 10⁻⁷ M.
 8. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said antibody or fragment or scaffold is not ADM-binding-Protein-1 (complement factor H).
 9. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said antibody or fragment or scaffold recognizes and binds to the N-terminal end (aa1) of adrenomedullin.
 10. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said antibody or said fragment or scaffold is an ADM stabilizing antibody or fragment or scaffold that enhances the half life (t1/2 half retention time) of adrenomedullin in serum, blood, plasma at least 10%, preferably at least 50%, more preferably >50%, most preferably >100%.
 11. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said antibody or fragment or scaffold blocks the ADM bioactivity not more than 80%, preferably not more than 50%.
 12. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said disease is selected from the group comprising sepsis, diabetes, cancer, heart failure, and shock.
 13. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said disease is not SIRS, sepsis, or septic shock.
 14. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said antibody or fragment is a human monoclonal antibody or fragment that binds to ADM or an antibody fragment thereof wherein the heavy chain comprises the sequences SEQ ID NO: 1 GYTFSRYW SEQ ID NO: 2 ILPGSGST SEQ ID NO: 3 TEGYEYDGFDY

and wherein the light chain comprises the sequences SEQ ID NO: 4 QSIVYSNGNTY SEQ ID NO: 5 RVS SEQ ID NO: 6 FQGSHIPYT.


15. A human monospecific anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 14, wherein said antibody or fragment comprises the sequences: SEQ ID NO: 7 (AM-VH-C) QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGE ILPGSGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGY EYDGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 8 (AM-VH1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 9 (AM-VH2-E40) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGR ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 10 (AM-VH3-T26-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 11 (AM-VH4-T26-E40-E55) QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGE ILPGSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGY EYDGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKHHHHHH SEQ ID NO: 12 (AM-VL-C) DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPK LLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC SEQ ID NO: 13 (AM-VL1) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC SEQ ID NO: 14 (AM-VL2-E40) DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPR RLIYRVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC


16. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1, wherein said antibody or fragment or scaffold is a modulating antibody or fragment or scaffold that enhances the half life (t1/2 half retention time) of adrenomedullin in serum, blood, plasma at least 10%, preferably at least 50%, more preferably >50%, most preferably >100% and that blocks the ADM bioactivity not more than 80%, preferably not more than 50%.
 17. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim 1 to be used in combination with vasopressors e.g. catecholamine and/or fluids administered intravenously.
 18. Anti-Adrenomedullin (ADM) antibody or an anti-ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold binding to adrenomedullin for use in therapy of an acute disease or acute condition of a patient according to claim
 1. 19. Pharmaceutical formulation comprising an anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold for use in a therapy of an acute disease or acute condition of a patient according to claim
 1. 20. Pharmaceutical formulation for use in therapy of an acute disease or acute condition of a patient according to claim 19, wherein said pharmaceutical formulation is a solution, preferably a ready-to-use solution.
 21. Pharmaceutical formulation for use in therapy of an acute disease or acute condition of a patient according to claim 20, wherein said pharmaceutical formulation is in a freeze-dried state.
 22. Pharmaceutical formulation according to claim 19, wherein said pharmaceutical formulation is administered intra-muscular.
 23. Pharmaceutical formulation according to claim 19, wherein said pharmaceutical formulation is administered intra-vascular.
 24. Pharmaceutical formulation according to claim 19, wherein said pharmaceutical formulation is administered via infusion.
 25. Pharmaceutical formulation according to claim 19, wherein said pharmaceutical formulation is to be administered systemically to a patient for prevention or reduction of organ dysfunction or organ failure in a patient having an acute disease or acute condition.
 26. Pharmaceutical formulation according to claim 19, wherein said pharmaceutical formulation is to be administered systemically via infusion to a patient for prevention or reduction of organ dysfunction or organ failure in a patient having an acute disease or acute condition. 